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(Circulation. 1995;92:415-420.)
© 1995 American Heart Association, Inc.

Articles

Human Heart–Infiltrating T-Cell Clones From Rheumatic Heart Disease Patients Recognize Both Streptococcal and Cardiac Proteins

L. Guilherme, PhD; E. Cunha-Neto, MD, PhD; V. Coelho, MD, PhD; R. Snitcowsky, MD; P. M. A. Pomerantzeff, MD; R. V. Assis, MD; F. Pedra, BS; J. Neumann, MD; A. Goldberg, PhD; M. E. Patarroyo, MD; F. Pileggi, MD; J. Kalil, MD, PhD

From the Instituto do Coração do Hospital das Clinicas, Faculdade de Medicina da Universidade de São Paulo (Brazil), and the Instituto de Immunologia, Hospital San Juan de Dios, Universidad Nacional de Colombia, Bogota, Colombia (M.E.P.).

Correspondence to Jorge Kalil, MD, PhD, Laboratório de Imunologia de Transplantes, Instituto do Coração, Faculdade de Medicina USP, Av Dr Eneas de Carvalho Aguiar, 500-3° Andar, 05403-000 São Paulo, Brazil.

	Abstract

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Background ß-Hemolytic streptococcal infection in developing countries still causes thousands of cases of rheumatic heart disease, demanding surgical valve correction. Antigenic mimicry between self and streptococcal components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic susceptibility. Although heart streptococcal–M protein cross-reactive antibodies have been demonstrated, heart tissue damage seems to be T lymphocyte–dependent. We studied the infiltrating T lymphocytes in rheumatic heart lesions with the aim of understanding the role of cellular immune response at the site of the lesions.

Methods and Results We obtained 107 T-cell clones from surgical fragments of cardiac tissue from four rheumatic heart disease patients. We tested their capacity to recognize streptococcal M protein–derived synthetic peptides and heart proteins. We found eight infiltrating T-cell clones from all four patients that simultaneously recognize streptococcal M and heart proteins. Among the M-protein sequences tested, only synthetic peptides corresponding to regions 1 through 25, 81 through 103, and 163 through 177 were simultaneously recognized with heart protein fractions. Interestingly, regions 81 through 103 and 163 through 177 have been known to bear heart cross-reactive epitopes at the antibody level. Five of these clones are CD4⁺, and one is CD8⁺.

Conclusions The presence of heart–M protein cross-reactive T-cell clones in rheumatic heart lesions suggests their direct involvement in the pathogenesis of this disease. The dissection of protective and pathogenic epitopes of streptococcal M protein is an important step in allowing the development of a safe anti-streptococcal synthetic vaccine.

Key Words: autoimmunity • rheumatic heart disease • T cell clones • molecular mimicry • group A ß-hemolytic streptococci

	Introduction

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Rheumatic fever (RF) is still a major public health problem in developing countries. In Brazil, for instance, valve correction owing to sequelae of rheumatic heart disease (RHD) corresponds to one third of the heart surgeries (data from the Ministry of Health). Although this disease is no longer considered a public health problem in developed countries where living standards in target populations were improved, epidemic episodes have recently been reported in the United States.¹

ß-Hemolytic streptococcal pharyngitis may induce RF and RHD in susceptible individuals weeks or years after acute infection episodes. Clinical features of RF present a great variation; RHD is a frequent and severe complication. Immunologic response is directed primarily against M protein, a major component of the streptococcal cell surface that presents antiphagocytic properties. M protein shares structural homology and antibody cross-reactivity with "-helical coiled-coil" fibrous proteins like myosin and tropomyosin in the heart muscle.^{2 3 4 5} Although most patients exhibit cross-reactive antibodies, these antibodies do not seem to contribute to tissue damage. The presence of CD4⁺ T cells at lesion sites in the heart has been demonstrated, suggesting a direct role for these cells in the pathogenesis of RHD.^{6 7} Furthermore, in favor of an important role for T cells in RF, M protein–stimulated T cells derived from the peripheral blood can display cytotoxic activity toward immortalized human heart cells.^{8 9} More recently, it was shown that streptococcal M protein may be a superantigen, ie, a protein capable of stimulating a large number of T cells that share a T-cell antigen receptor variable region element,^{10 11 12} a fact that can have important immunopathological consequences.

Several recent studies have approached the antigen recognition repertoire of tissue-infiltrating T cells in autoimmune diseases by establishing functional lines and clones from affected tissue.^{13 14 15} To the best of our knowledge, this is the most direct approach for identification of relevant antigenic targets in vivo. We have thus derived T-cell lines that were subsequently cloned from myocardium and mitral and aortic valve surgical fragments of four RHD patients.

	Methods

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Patients
We studied four patients with cardiac failure attending the Heart Institute and submitted to surgery for valve correction. All patients (S.R.S., S.L.A., W.F.A., and F.F.S.) were children (mean age, 11 years) predominantly with mitral valve lesions. Two patients (S.L.A. and W.F.A.) had had recent episodes of disease activity.

T-Cell Lines and Clones
T-cell lines were generated from "in vitro" culture of surgical fragments of mitral valve, papillar muscle, and left atrium from four RHD patients. Tissue was finely minced with injection needles and small scissors and placed in Falcon flat-bottom 96-well plates (Becton, Dickinson & Co) with Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical Co) supplemented with 2 mmol/L L-glutamine (Sigma), 10% pooled normal human serum, 10 mmol/L HEPES (Sigma), antibiotics (Gentamycin and Peflacyn) at 40-µg/mL concentration, and 40 U/mL of human recombinant interleukin-2 (Biosource Inc) on an HLA-DR–matched feeder layer of peripheral blood mononuclear cells (PBMC, 1x10⁵ cells per well) irradiated at 5000 rad.^{16 17}

T-cell clones were obtained by the limiting dilution method in the presence of 10 µg/mL PHA-P (Sigma), 1x10⁵ irradiated PBMC per well in interleukin-2–enriched growth medium as above. Plates that had more than 15% positive wells were discarded.

We used the same protocol to derive some clones from endomyocardial biopsies from six patients with other heart inflammatory conditions (three patients with chronic Chagas' cardiomyopathy and three heart transplant patients undergoing allograft rejection) as negative control of the experiments.

Immunohistochemistry
Sections (4 µm) were cut from cardiac tissue prepared from frozen surgical fragments and specimens embedded in OCT 4583 (Miles Laboratories Inc). Anti-CD3, anti-CD4, anti-CD8 (all from Dakopatts), anti–TCR 1 (T Cell Diagnostics, Inc) monoclonal antibodies were used to define T-cell subpopulations. Peroxidase-coupled avidin (Dakopatts) was added later, and the reaction was developed with diaminobenzidine (Sigma).

Flow Cytometry analysis
T-cell lines and clones were analyzed with the same anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies used for immunohistochemical analysis, with phycoerythrin-labeled goat anti-mouse IgG (IG-R-PE, Southern Biotechnology Associates, Inc) as secondary antibody. T cells were detected with fluorescein isothiocyanate–labeled anti–TCR 1 antibody (T Cell Diagnostics).

Peptide Synthesis and Preparation of Heart Tissue Protein Fractions
Peptides based on the published M protein type 5 sequence (M5)^{18 19} were synthesized by the "tea bag" method²⁰ and then purified by high-performance liquid chromatography.²¹ Heart tissue fractions were obtained from lysates of postmortem normal human myocardium and aortic valve tissue, separated by SDS-PAGE, and blotted onto nitrocellulose membranes.²² The blots were divided into several horizontal strips with approximately the same amount of protein. A nitrocellulose strip without protein was used as negative control. The strips were solubilized in dimethyl sulfoxide (E. Merck) reprecipitated in sodium carbonate/sodium bicarbonate buffer 0.05 mol/L, pH 9.6, and washed with RPMI 1640 medium (Sigma), yielding a fine suspension of protein-loaded nitrocellulose particles. Fig 1 shows the amino acid sequence of peptides and the molecular weight range of each heart tissue protein fraction.

View larger version (25K): [in this window] [in a new window]   Figure 1. Chart showing nine peptides based on the M protein type 5 sequence: 307, residues 1 through 20; 308, residues 11 through 25; 309, residues 62 through 82; 327, residues 81 through 96; 326, residues 83 through 103; 310, residues 91 through 103; 311, residues 125 through 139; 312, residues 141 through 154; and 313, residues 163 through 177. Molecular weight is from seven myocardium protein fractions and six aortic valve protein fractions.

Proliferation Assays
Proliferation assays were performed in Falcon flat-bottom 96-well plates using 2x10⁴ cloned cells per well with 1x10⁵ HLA-DR–matched irradiated PBMC (5000 rad) for 72 hours at 37°C in a humidified CO₂ incubator. Peptides were used in either monomeric or polymeric form²³ at 1 µg/mL and 20 µL per well of heart tissue fractions, as previously determined by titration. Negative controls were DMEM with feeder for the peptide experiments and 20 µL of a protein-free nitrocellulose suspension for heart tissue fraction experiments. PHA-P (10 µg/mL) and anti-CD3 (200 ng/mL) (data not shown) were positive controls for proliferative responses. Triplicate wells were pulse-labeled with 1 µCi per well of tritiated thymidine (Amersham Life Sciences) for the final 18 hours of culture, harvested, and analyzed in a scintillation ß-counter. The proliferative response of T-cell clones was considered positive when Student's t test probability value was <.01. The stimulation index (SI, mean experimental counts per minute per negative control counts per minute) was considered significant when >2.5.

	Results

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We obtained 107 clones from surgical fragments of four RHD patients (Table 1) that were tested for proliferation against nine synthetic peptides derived from the published sequence for streptococcal M5, seven electrophoretic protein fractions from normal human myocardium, and six electrophoretic protein fractions from normal human aortic valve (Fig 1).

View this table: [in this window] [in a new window]   Table 1. Identification of T-Cell Clones

Among the 107 clones tested, 8 (7.5%) recognized both an M5 synthetic peptide and a heart protein fraction (Table 1). These cross-reactive clones recognized three different regions of the M5 molecule. Two clones reacted against residues 1 through 25 (Fig 2a and 2b). Three clones recognized the region corresponding to amino acid residues 163 through 177 (Fig 2c, 2e, and 2f). Finally, 3 clones recognized the amino acid sequence 81 through 103 (Figs 2d, 2g, and 3). Of 8 cross-reactive T-cell clones, 6 recognized aortic valve tissue fractions; 5 of 8 cross-reactive T-cell clones recognized aortic valve fraction II (90 to 150 kD) or V (30 to 43 kD); 4 of 8 recognized aortic valve fraction IV (43 to 65 kD). Aortic valve fractions I (>150 kD), III (65 to 90 kD), and VI (10 to 30 kD) were recognized only once. Interestingly, aortic valve fractions II and V were simultaneously recognized by 4 of 8 T-cell clones. Myocardium fractions were recognized by 4 of 8 cross-reactive T-cell clones; fractions I (>150 kD) and VI (24 to 30 kD) by 2 of 8 clones. Fractions II (95 to 150 kD), III (65 to 95 kD), and IV (44 to 65 kD) were recognized only once.

View larger version (24K): [in this window] [in a new window]   Figure 2. Bar graphs showing positive proliferative response of seven clones to M protein type 5 peptides (Pep), myocardium (Myoc Fr), and aortic valve fractions (Aort v Fr). Results are shown as stimulation index (SI). Counts per minute of negative controls is shown (Pep NC and Fr NC).

View larger version (20K): [in this window] [in a new window]   Figure 3. Bar graphs showing proliferative response to M protein type 5 (A) peptides, shown as stimulation index (SI) (A). Negative control, 116 cpm; positive response for peptide 326 (SI=4.9). B, Proliferative response to heart tissue fractions shown as SI. Negative control, 148 cpm; positive response for myocardium fraction I (SI=3.3) and aortic valve fractions II (SI=3.9), IV (SI=4.7), and V (SI=4.0). indicates myocardium fraction; , aortic valve fractions.

We also tested the recognition of the same set of M5 synthetic peptides by 42 T-cell clones derived with the same protocol from endomyocardial biopsies of unrelated heart inflammatory lesions, Chagas' disease cardiopathy, and heart allograft rejection. None of the clones derived from biopsies of patients with chronic Chagas' cardiomyopathy (22 clones from three individuals) and heart transplant patients undergoing allograft rejection (20 clones from three individuals) recognized any of the tested M5 synthetic peptides, presenting SI 1.5. Heart tissue fractions were not tested with these clones.

Immunohistochemical staining of heart tissue from the same patients (Table 2) showed the predominance of infiltrating CD4⁺ T cells. Heart-derived T-cell lines expressed either the CD3⁺CD4⁺CD8^- or CD3⁺CD4^-CD8⁺ phenotype. In one patient, we found a T-cell line in which most of the cells were TCR ⁺ (Table 2).

View this table: [in this window] [in a new window]   Table 2. T-Cell Subsets

Of 6 cross-reactive clones, 5 were CD4⁺; nevertheless, 1 cross-reactive clone (Lu1.1.27) displayed a CD8⁺ phenotype (Table 2). T-cell clones Lu 1.1.2 and Lu 7.1.9 were not tested.

	Discussion

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We have derived for the first time T-cell clones from heart lesions of RHD patients. Furthermore, some of these T-cell clones recognize both streptococcal M-protein synthetic peptides and heart tissue protein fractions. The availability of heart valve tissue after heart surgery in RHD patients has allowed close investigation of the functional role of heart–infiltrating T cells in the disease process. The evidence for antibody cross-reactivity between streptococcal M protein and structurally related heart proteins and the DTH-like T-cell infiltrate rich in CD4⁺ T cells and macrophages^{6 7} seen in the site of lesions pointed toward the existence of disease-inducing streptococcal M protein–heart tissue cross-reactive T cells in situ. The immunohistochemical analysis confirmed the predominance of CD4⁺ T lymphocytes in our samples. However, the phenotypes expressed by the heart-derived T-cell lines (Table 2) showed that different T-cell populations could grow under our culture conditions.

We have studied antigen-specific T-cell responses to streptococcal M5 and heart tissue protein fractions for three reasons. First, we have evidence for molecular mimicry between streptococcal M protein and heart proteins. Second, M5 was chosen because Streptococcus pyogenes type M5 is the most frequent isolate from RF patients.²⁴ Third, because no heart proteins cross-reactive at the human T-cell level with M protein have been previously established, we have chosen to use heart protein fractions²² instead of purifying the major heart proteins.

Among the 107 clones tested, 8 (7.5%) recognized both an M5 synthetic peptide and a heart protein fraction (Table 1). Cross-reactive T-cell clones were obtained from all four patients regardless of the source of the heart tissue fragment cultured. Five cross-reactive clones were CD4⁺ (Table 2), which is consistent with the available histopathological data indicating that delayed-type hypersensitivity is the major mechanism leading to tissue damage in RHD.²⁵ Nevertheless, 1 cross-reactive clone (Lu1.1.27) displayed a CD8⁺ phenotype. This finding is intriguing because antigen presenting cells in the assay were selected for HLA-DR matching.

Heart protein cross-reactive recognition involved a limited number of epitopes of the M5 molecule: the 1 through 25, 81 through 103, and 163 through 177 amino acid residues (Figs 2 and 3). These epitopes were recognized by heart-recognizing T-cell clones from one, three, and two patients, respectively (Figs 2 and 3). The 1 through 25 sequence of M5 used to be accounted for as a protective anti–S. pyogenes epitope.²⁶ The 81 through 103 region is known to present cross-reactivity with cardiac myosin at the antibody level in mice, rabbits, and humans.^{3 5 27} The 163 through 177 region has been shown to display antibody cross-reactivity with sarcolemmal antigens in M protein–immunized rabbits.^{2 5} It is interesting to note that, while synthetic peptides encompassing other regions of streptococcal M5 were occasionally recognized by 17 of 107 tissue-infiltrating T-cell clones, such clones never displayed cross-reactivity with heart protein fractions (data not shown). The selectivity in cross-reactive recognition of the three discrete M5 regions by heart-derived T-cell clones from HLA-disparate individuals is suggestive of a limited number of heart cross-reactive epitopes in M5 protein. The frequent heart cross-reactive recognition of M5 regions 81 through 103 and 163 through 177 may indicate that these regions might be common targets of antigenic mimicry.

Among mice immunized with recombinant M5, T-cell clones were derived against six different synthetic peptides of the NH₂ terminal sequence.²⁸ It is interesting to note that only one epitope was recognized simultaneously by murine T-cell clones (residues 1 through 35) and the human clones reported here (residues 1 through 25).

Most T-cell clones simultaneously recognized several protein fractions derived from either myocardium or aortic valve (Figs 2 and 3). The predominant recognition of heart valve tissue fractions is consistent with the more prevalent and extensive damage to heart valves than to myocardial tissue. The recognition of multiple tissue fractions by a given T-cell clone may be due to two mechanisms: the recognition of similar epitopes in structurally similar -helical coiled-coil protein in a different protein fraction and the existence of protein degradation during the preparation of protein fractions, reflected in the presence of a higher-molecular-weight component in lower-molecular-weight fractions.²⁹

Although most studies demonstrated cross-reactive antibodies between streptococcal M protein and heart myosin, the antigenic mimicry may be directed toward -helical coiled-coil structural domains shared by many proteins.³⁰ In fact, monoclonal antibodies derived against M protein and cross-reactive to myosin are also reactive against tropomyosin, keratin, laminin, and vimentin; one such cross-reactive antibody recognized a 116-kD protein in heart valve Western blots.³¹ It is possible that the frequent (5 of 8) cross-reactive T-cell recognition of heart protein fraction II (95 to 150 kD) is due to the cross-recognition of M-protein peptides and the 116-kD component described in antibody studies. Fraction IV (43 to 65 kD), recognized by 4 of 8 M protein–cross-reactive T-cell clones, may contain vimentin, the 57-kD major valve fibroblast antigen described in M protein–cross-reactive antibody studies. Some of our T-cell clones (5 of 8) consistently recognized two bands, the aortic valve fractions II and V (Figs 2 and 3). Studies to characterize the nature of the primary stimulatory heart proteins present in the cross-reactive fractions are in progress.

Previous studies in our laboratory and work by others identified the association of susceptibility to RF to several different HLA class II specificities.^{32 33 34 35 36 37 38} We hypothesize that the several HLA specificities described in association with RF may present M-protein peptides that elicit recognition of tissue components. The fact that patients displaying cross-reactive T-cell clones bear different HLA class II alleles suggests that the cross-reactive M5 peptides may bind to several different alleles sharing conformational motifs in the peptide-binding groove.^{39 40}

The puzzling finding that streptococcal M protein can induce T-cell blastogenesis from normal noninfected humans^{8 9} was further explained when it was found that streptococcal M protein may be a superantigen stimulating mostly TCR Vß1, 2, 4, 5.2, and 8.^{10 11 12} The possibility that a subset of cross-reactive M protein–heart protein–specific T-cell clones can bear the required TCR Vß element, thus undergoing massive amplification in the presence of the streptococcal protein, may be the underlying reason for the common relapses of RHD after streptococcal reinfections. The TCR V/Vß profile of the tissue-infiltrating T-cell clones described here is currently under investigation.

To the best of our knowledge, this is the first report of human infiltrating T-cell cross-reactivity to a bacterial product and to human tissue in established postinfectious autoimmune disease. We could establish the significance of molecular mimicry between ß-hemolytic streptococci and heart tissue, assessing the T-cell repertoire leading to heart tissue damage in RHD. It is noteworthy that aortic valve fractions were recognized more frequently than myocardium. In fact, the evolution of the disease shows that after pancarditis, the damage is directed primarily to heart valves. The fact that none of the 42 T-cell clones derived from biopsies of patients with chronic Chagas' cardiomyopathy and heart transplant patients undergoing allograft rejection recognized any of the tested M5 synthetic peptides reinforces the specificity and relevance of cross-reactive recognition by heart-derived T-cell clones in all four RHD patients tested. Although this study does not address the actual role of T cells in tissue damage in RHD, the identification of "relevant" antigen recognition is certainly consistent with such a role. The definition of the relevant antigens in a postinfectious autoimmune disease may allow the use of antigen-specific immunosuppression approaches to ablate the deleterious autoimmune response without interfering with antipathogen immunity. In addition, it may finally allow the search for effective subunit vaccines devoid of components of S. pyogenes that bear the pathogenic heart cross-reactive determinants leading to RHD.

	Acknowledgments

 
This work was supported by grants from the PADCT-CNPq No. 62.0418/91.5 and FAPESP 90/2885-6. We express our gratitude to Prof Dr Luiz V. Décourt for helpful discussions.

Received September 26, 1994; revision received January 10, 1995; accepted January 19, 1995.

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