(Circulation. 1995;92:415-420.)
© 1995 American Heart Association, Inc.
Articles |
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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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 proteinderived 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 heartM 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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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
proteinstimulated 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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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-DRmatched 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-2enriched 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),
antiTCR
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
isothiocyanatelabeled antiTCR
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.
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Proliferation Assays
Proliferation assays were performed in
Falcon flat-bottom
96-well plates using 2x104 cloned cells per well with
1x105 HLA-DRmatched 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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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.
|
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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
).
|
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 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 antiS. 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 proteinimmunized
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 proteincross-reactive T-cell clones, may
contain vimentin, the 57-kD major valve fibroblast antigen described in
M proteincross-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 humans8 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
proteinheart proteinspecific 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 |
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Received September 26, 1994; revision received January 10, 1995; accepted January 19, 1995.
| References |
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