Molecular evidence for antigen-driven immune responses in cardiac lesions of rheumatic heart disease patients

Luiza Guilherme1, Nicolas Dulphy5, Corinne Douay5, Verônica Coelho1, Edécio Cunha-Neto1, Sandra E. Oshiro1, Raimunda V Assis1,4, Ana C. Tanaka1, Pablo M. Alberto Pomerantzeff1, Dominique Charron5, Antoine Toubert5 and Jorge Kalil1,2,3

1 Heart Institute–InCor, University of São Paulo, School of Medicine, and
2 Clinical Immunology and Allergy, Department of Clinical Medicine, and
3 International Scholar Howard Hughes Medical Institute, São Paulo, Brazil
4 University of Juiz de Fora, Department of Pathology, Minas Gerais, Brazil
5 Laboratoire d'Immunologie et d'Histocompatibilité, INSERM U396, Institut Universitaire d'Hématologie, Hôpital St Louis, Paris, France

Correspondence to: L. Guilherme, Laboratório de Imunologia de Transplantes, Instituto do Coração, HC-FMUSP, Av. Dr Eneas de Carvalho Aguiar, 500–3° andar 05403-000, São Paulo, SP, Brazil


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatic heart disease (RHD) is a sequel of post-streptococcal throat infection. Molecular mimicry between streptococcal and heart components has been proposed as the triggering factor of the disease, and CD4+ T cells have been found predominantly at pathological sites in the heart of RHD patients. These infiltrating T cells are able to recognize streptococcal M protein peptides, involving mainly 1–25, 81–103 and 163–177 N-terminal amino acids residues. In the present work we focused on the TCR ß chain family (TCR BV) usage and the degree of clonality assessed by ß chain complementarity-determining region (CDR)-3 length analysis. We have shown that in chronic RHD patients, TCR BV usage in peripheral blood mononuclear cells (PBMC) paired with heart-infiltrating T cell lines (HIL) is not suggestive of a superantigen effect. Oligoclonal T cell expansions were more frequently observed in HIL than in PBMC. Some major BV expansions were shared between the mitral valve (Miv) and left atrium (LA) T cell lines, but an in-depth analysis of BJ segments usage in these shared expansions as well as nucleotide sequencing of the CDR3 regions suggested that different antigenic peptides could be predominantly recognized in the Miv and the myocardium. Since different antigenic proteins probably are constitutively represented in myocardium and valvular tissue, these findings could suggest a differential epitope recognition at the two lesional heart sites after a common initial bacterial challenge.

Keywords: autoimmunity, M protein, rheumatic heart disease, superantigens, TCR


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatic fever (RF) is a consequence of throat infection by ß-hemolytic group A streptococci, affecting 3–4% of non-treated children and therefore a major health problem in Brazil. Rheumatic heart disease (RHD) develops 4–8 weeks or later after streptococcal infection in ~30% of individuals with RF.

The pathogenic mechanisms involved in the development of RF/RHD remain unclear; however, it is evident that an abnormal humoral and cellular immune response occurs. Antigenic mimicry between streptococcal antigens, mainly M protein epitopes, and heart components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic predisposition (13). Several genetic markers of susceptibility were studied and no consistent association was found (4); however, associations with different HLA class II antigens have been observed in several populations (513). Since HLA class II antigens play an important role in the antigen presentation to the TCR, the variable association with HLA antigens is consistent with the possibility that different serotypes of group A streptococci could be implicated in the disease in different countries. M proteins are the major targets of the host anti-streptococcal immune response (14). The heart is considered as an immunocompetent organ with lymph node drainage, and is consequently under immune surveillance by lymphocytes and macrophages. Dendritic cells expressing HLA class I and class II molecules at their surface and able to present antigens to T lymphocytes have been described in the heart (15). In acute RF, Aschoff bodies (conglomerates of monocytes/macrophages and neutrophils) are frequently found and play an important role in the triggering of a local inflammatory process, acting as antigen-presenting cells (16,17). Autoreactivity to heart antigens caused by microbial infections was described in several heart diseases (1820). Superantigens are proteins derived from bacteria and viruses that polyclonally activate T cells by an MHC class-II dependent, but haplotype-unrestricted, mechanism (2123). Proliferative responses to superantigens are limited to T cells expressing particular TCR BV gene families and are independent of antigen specificity. Superantigens are known to activate autoreactive T cells and initiate an inflammatory process leading to autoimmunity in animal models, although a definite role for superantigens in the pathogenesis of human autoimmune diseases is still a matter of debate (24). Several streptococcal pyrogenic exotoxins, but not M5 protein, display superantigenic properties (2527).

The TCR ß chain is produced by the assembly of variable (V)–diversity (D)–joining (J) and constant (C) gene segments. The V–D–J junction encodes the hypervariable region of the TCR designated as complementarity-determining region (CDR)-3 that interacts directly with the complex peptide–MHC molecule. Two methods called Spectratyping and Immunoscopy have been described (28,29) in order to determine the size of CDR3 regions in transcripts of whole BV families or in BV–BJ combinations, and are particularly suitable for analysis of {alpha}ß T cell clonality in tissue-infiltrating T cells (30). Patterns of ß chain CDR3 diversity are definitely different in the case of activation by superantigens or nominal antigens giving rise respectively to polyclonal expansions of whole BV families or to the disturbance of the Gaussian distribution of CDR3 size by one or several oligoclonal expansions (31).

The presence of CD4+ T cells in the heart of RHD patients has been demonstrated, suggesting a direct role for these cells in the pathogenesis of RHD (32). In a previous work, we showed that T lymphocyte clones infiltrating the heart lesions of severe RHD patients are able to recognize myocardium and valve proteins as well as immunodominant streptococcal M5 peptides, a finding suggestive of molecular mimicry at the T cell level between ß hemolytic streptococci and heart tissue (33). The aim of the present work was to compare the TCR BV usage in peripheral blood and heart-infiltrating T cell lines (HIL) from RHD patients, looking for oligoclonal ß chain expansions in line with antigen-driven immune responses, and for a possible superantigenic effect still detectable at chronic RHD patients. We also wanted to evaluate the complexity of this response at different pathological sites in the same and in different patients by determining the ß chain CDR3 size profile, and by defining the BJ segment usage and the amino acid sequences of selected T cell expansions shared at different sites in the heart.


    Methods
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 Methods
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 References
 
Patient samples
Peripheral blood mononuclear cells (PBMC) were obtained from six patients with severe RHD, from the Heart Institute HC–FMUSP, selected according to Jones' modified criteria (34). The average length of patient follow-up was 5 years. Blood samples were taken in the absence of immunosuppressive drugs. Patient data and samples used in TCR studies are summarized in Table 1Go. Both blood samples and surgical fragment collection procedures were cleared by the Committee of Ethics of the Heart Institute, HC–FMUSP.


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Table 1. Identification and HLA-DR/DQ of patients, clinical and histopathology characteristics and samples analysed for TCR studies
 
PBMC and T cell lines
PBMC were obtained by density gradient centrifugation (Ficoll-Hypaque) and were isolated directly by three successive washes in PBS. Heart-infiltrating T cell lines (HIL) were derived from surgical heart fragments obtained during valve correction in four of these patients. Two patients (WFA and JSS) yielded two T cell lines, derived from mitral valve (Miv) and left atrium (LA). For patients SLA and JEB T cell lines were derived from Miv and LA respectively. Tissue was finely minced with injection needles and small scissors, placed in Falcon flat-bottom 96 multi-well plates (Becton Dickinson, Lincoln Park, NJ), with DMEM (Sigma, St Louis, MO) supplemented with 2 mM L-glutamine (Sigma), 10% pooled normal human serum, 10 mM HEPES (Sigma), antibiotics (Gentamycin and Peflacyn) at the concentration of 40 and 20 µg/ml respectively, and 40 U/ml of human recombinant IL-2 (Biosource, Camarillo, CA), on a HLA-DR matched feeder layer of PBMC at 105 cells/well, irradiated at 5000 rad (35,36).

HLA typing
Patients were typed for HLA-DR and -DQ by PCR reactions. Briefly, 24 PCR reactions were performed per patient, 20 for assigning HLA-DR1 to -DR18, one each for HLA-DR51, -DR52 and -DR53, and one amplification control. For DQ1 to DQ9 specificities, eight different PCR reactions were sufficient (37).

Immunohistochemistry
Sections of 4 µm were cut from cardiac tissue prepared from frozen surgical fragments and specimens embedded in OCT 4583 (Miles, Naperville, IL). Anti-CD4 (MT 310) and CD8 (DK 25) (Dakopatts, Glostrup, Denmark) mAb were used to define T cell subpopulations. Peroxidase-coupled avidin (Dakopatts) was added later and the reaction was developed with diaminobenzidine (Sigma).

FACS
T cell subpopulations from intralesional T cell lines were analyzed using anti-{alpha}ß TCR–FITC (T10B9.1A-31) (PharMingen, San Diego, CA), anti-CD3–FITC (UCTH1), CD4–phycoerythrin (MT310) and CD8–FITC (DK 25) mAb (Dakopatts). In total, 5x103 events gated on lymphocytes were analyzed using a FACScan flow cytometer with CellQuest software (Becton Dickinson, Mountain View, CA).

RNA extraction and cDNA synthesis
RNA was extracted from cell pellets of intralesional T cell lines (8–10 days after the first or second phytohemagglutinin stimulation in the presence of IL-2 and feeder cells) and PBMC by lysis in guanidium thiocyanate buffer. RNA extracted from paired wells containing 5x106 irradiated feeder cells, PHA, IL-2 and incubated for the same 8–10 days was undetectable. The PBMC and HIL cDNA were prepared from 1–10 µg total RNA with AMV reverse transcriptase (cDNA cycle kit; Invitrogen, Leek, The Netherlands) as described (38).

Oligonucleotides and CDR3 size analysis
The nomenclature of BV families and the primers used have been described (38,39). Fluorescent primers for BC and BJ were labeled at the 5' end with the Fam fluorophore (Applied Biosystems, Foster City, CA). Aliquots of the cDNA synthesis reaction (corresponding to 250 ng of total RNA) were amplified in 50 µl reactions with one of the BV-specific oligonucleotides as the 5' primer and the BC oligonucleotide as the 3' primer. The final concentration was 0.5 mM for each primer, dNTP 0.2 mM, MgCl2 2 mM in Taq polymerase buffer (Promega, Madison, WI) in the presence of 1 U of Taq polymerase (Promega) on a DNA thermal cycler (9600; Perkin-Elmer, Norwalk, CT). The PCR cycle profile was : denaturation at 94°C for 30 s, annealing at 60°C for 45 s, primer extension at 72°C for 45 s for 40 cycles and a final polymerization step of 5 min at 72°C. Aliquots from each BV–BC PCR product (2 µl) were copied in six-cycle run-off reactions primed with a fluorophore-labeled BC or BJ oligonucleotide. The final concentration of dNTP was 0.2 mM, 3 mM MgCl2 in the presence of 0.2 U of Taq polymerase. The run-off reactions were migrated on 4.25% acrylamide sequencing gels (377A DNA sequencer; Applied Biosystems) for size (Genescan-500 size marker; Perkin-Elmer) and fluorescence intensity determination. The raw data were analyzed with the help of the Immunoscope software (29). The CDR3 region was defined to include residues 95–106 (30). Since the positions of the BV and the BC primers are fixed, the length distribution observed in the PCR fluorescent BV–BC products depends only on the size of the V–D–J junctions. Statistical analysis was performed to determine whether or not a profile could be considered as Gaussian: a profile was not considered to be Gaussian if one peak was excluded from the 95% confidence interval of peak level intensities. TCR B subfamilies BV10 and BV19 were omitted from this analysis as they are pseudogenes in most individuals (40).

BV and BJ gene usage
PBMC cDNA samples were quantified as described (38), and ~3x106 copies of cDNA from each PBMC sample were then amplified for 30 cycles with the BV primers and an internal fluorescent BC primer. BJ usage was defined only for some intralesional T cells expressing oligoclonal BV families after run-off reactions of the unlabeled BV–BC amplification product and is quantitative since the fluorescent primers have comparable amplification efficiencies (30). The fluorescence intensity in each BV or BJ family was expressed as the percentage of total signals from the 22 BV or 13 BJ subfamilies.

DNA sequencing
BVBJ PCR products were cloned into pCR®2.1 vector (Invitrogen) and transformed into XL1 Blue supercompetent cells (Stratagene, La Jolla, CA). After blue/white screening of recombinant plasmids on X-galactoside/isopropylthiogalactoside (IPTG) indicator plates, plasmids were purified by alkaline lysis followed by phenol–chloroform–iso-amyl alcohol. Inserts were checked by agarose gel electrophoresis after BV–BJ PCR amplification and both strands were sequenced with the ABI Prism Dye Primer Cycle sequencing kit (Perkin-Elmer). Products were loaded on 4.25% acrylamide sequencing gels (377A DNA sequencer; Applied Biosystems) and analyzed with the Sequence Navigator software.

Peptide synthesis and preparation of human heart tissue protein fractions
Peptides based on the published M5 protein sequence (41,42) were synthesized by the `tea bag' method by t-BOC chemistry (43), and were checked by mass spectrometry and HPLC. Tissue fractions from human ventricular myocardium, aortic valve, were obtained from lysates of post-mortem normal tissue samples, separated by SDS–PAGE and blotted onto nitrocellulose membranes (Sigma) (44,45). The blots were divided in several horizontal strips with approximately the same amount of protein with defined mol. wt values as previously described (33). The strips were solubilized in DMSO (Merck, Darmstadt, Germany), precipitated in sodium carbonate/sodium bicarbonate buffer 0.05 M, pH 9.6, and washed with RPMI 1640 medium (Sigma, St Louis, MO) to yield a fine suspension of protein-loaded nitrocellulose (see Table 4Go for peptides sequences and heart proteins mol. wt).


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Table 4. Reactivity of intralesional T cell lines and peripheral blood T cells from severe RHD patients against immunodominant streptococcal M5 protein region and heart tissue protein fractions
 
Proliferation assays
Proliferation assays were performed in Falcon flat-bottom 96 multiwell plates (Becton Dickinson, Lincoln Park, NJ), using 105 mononuclear cells/well isolated from peripheral blood by centrifugation on a d = 1077 density gradient, with 5 µg/ml of streptococcal M5 peptides or 20 µl/well of heart tissue fractions added for 96 h at 37°C in a humidified 5% CO2 incubator. Negative controls were mononuclear cells in DMEM (Sigma) without antigens for the peptide experiments and 20 µl of a protein-free nitrocellulose suspension for heart-tissue fraction experiments. PHA-P (5 µg/ml) (Sigma) was used for positive control of proliferative responses. Triplicate wells were pulse-labeled with 1 µCi/well of [3H]thymidine (Amersham Life Sciences, London, UK) for the final 18 h of culture; cells were harvested and analysed in a automated gas-phase ß-counter (Matrix 96; Packard, Camberra, Australia). For T cell lines we used 2x104 T cells/well with 105 HLA-DR-matched irradiated PBMC (5000 rad) for 72 h. The proliferative response was considered positive when the Stimulation Index (SI = mean experimental c.p.m./negative control c.p.m.) was >=2.5.


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T cell subsets in heart tissue and intralesional T cell lines
Immunohistochemical determination was performed on heart tissue fragments of three patients (SLA, WFA and JSS) and CD4+ T cells were predominantly found (Table 2Go). Six intralesional T cell lines were generated from Miv and LA (myocardium) fragments. All T cell lines were predominantly CD3+ and {alpha}ß TCR+ and most (five of six) were also predominantly CD4+ (Table 2Go).


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Table 2. Surface phenotype of T cells from surgical samples and T cell lines derived in vitro
 
Level of expression of TCR BV gene in PBMC and intralesional T cell lines
We measured by a semiquantitative analysis the frequency of 22 TCR BV families in PBMC from six severe RHD patients and the intralesional T cell lines described in Tables 1 and 2GoGo. The relative frequency in both periphery and intralesional T cell cultures showed no particular expansion of any BV family. Some BV families (BV2, BV3, BV6 and BV22) (Fig. 1Go) were highly expressed in the periphery (>8.0% each) for most of the patients. In intralesional T cell cultures the most frequent BV families were BV2, BV3, BV6 and BV22 (Fig. 2Go). The overall pattern of BV gene expression was similar in PBMC versus HIL, but when compared in single individuals some BV families appeared to be more expressed among PBMC than in HIL or vice versa. For instance, in patient WFA, BV6 was more expressed in the periphery than in Miv HIL (15.0 versus 5.7% respectively) while BV15 was more highly expressed in myocardium-derived T cells (8.0 versus 1.0 and 1.5% in Miv-derived T cells and in the periphery respectively). There was no statistical difference in PBMC BV usage in RHD patients compared to normal individuals tested under identical technical procedures (38,46).



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Fig. 1. Repertoire of TCR BV families in PBMC of RHD patients. Family BV 24 was not detected. BV10 and BV19 are pseudogenes and therefore not represented.

 


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Fig. 2. Repertoire of TCR BV families in infiltrating T cell lines of RHD patients. BV 12 was not detected. BV10 and BV19 are pseudogenes and not represented.

 
CDR3 size patterns in TCR BV families
The analysis of TCR BV and BJ genes using the Immunoscope approach allows an evaluation of the T cell diversity in given BV families by analyzing CDR3 size patterns (29). The obtained BV–BC patterns were grouped according to their patterns into three groups as: oligoclonal profiles, one or two peaks above the Gaussian background and Gaussian profiles (Fig. 3Go). Our results showed that oligoclonal T cell expansions are found more frequently in HIL than in PBMC. Few oligoclonal expansions were found in the PBMC (BV8 and BV23 in SLA; BV7 and BV8 in JEB; BV18 in JSS; BV4, BV8 and BV15 in NCM and BV8 in ESS). Patient WFA presented no true oligoclonal expansion in PBMC with one or at most two peaks above the Gaussian background (Fig. 3Go).



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Fig. 3. Schematic summary of CDR3 size patterns from patients SLA, JEB, WFA and JSS in PBMC and intralesional T cell lines. Black rectangles correspond to oligoclonal profiles accounting for >40% of the total fluorescent signal in a given BV family. Dark rectangles correspond to one or two peaks above the Gaussian background accounting for 25–40% of the total fluorescence and light-gray rectangles to Gaussian profiles. White rectangles correspond to BV subfamilies not detected.

 
The CDR3 size distribution of major TCR BV expansions of PBMC and T cell lines of patients JEB, JSS, SLA and WFA are presented in Fig. 4Go. When we compared the patterns of the CDR3 size diversity in PBMC and intralesional T cell cultures we found several pictures: polyclonal families in both PBMC and intralesional T cell cultures (e.g. BV6 in patient JEB); oligoclonal BV expansions at one site only (BV22 in JSS Lu 4.2 and BV24 in JSS Lu 4.1; BV4 in WFA Lu 7.1 and BV13 in WFA Lu 7.2); oligoclonal expansions in PBMC and heart tissue either at the same size (BV4 in WFA PBMC and Lu 7.1) or not (BV13 in SLA PBMC and Lu 3.1). In other cases we found a polyclonal profile in PBMC and oligoclonal peaks in both LA and Miv T cell lines at a different (BV13 in WFA Lu 7.1 and Lu 7.2) or at the same CDR3 size (BV1 and BV23 in JSS; BV5 in WFA).



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Fig. 4. CDR3 size distribution of major TCRBV expansions in PBMC and HIL. Mitral valve-derived T cell lines (Lu 3.1, Lu 4.1 and Lu 7.1); LA T cell lines (Lu 5, Lu 4.2 and Lu 7.2). The 10 amino acids CDR3 size is indicated on the abcisses scale.

 
TCR BJ usage in expanded TCR BV families
The finding of oligoclonal expansions with the same CDR3 length in some BV families led us to look at the dominant BJ usage in major TCR BV expansions of HIL from JSS, WFA, SLA and JEB patients (Table 3Go). In some BV expansions we found a unique BJ segment, e.g. BJ2S3 in the BV1 expansion for patient SLA or BV17–BJ1S3 for patient JEB. In other cases several BJ segments accounted for the BV–BC major expansion as for instance in the BV23 expansions (10 amino acids) found in common in Miv and LA HIL of patient JSS. Notably, the multiple BJ segments were different in the two HIL from this patient. The patient WFA had also an oligoclonal BV5 expansion at a 10 amino acids CDR3 size in both HIL Lu 7.1 (Miv) and Lu 7.2 (LA). Conversely, in this case the same BJ2S3 gene segment was used in both intralesional T cell lines (Table 3Go and Fig. 5Go).


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Table 3. BJ gene segments in relevant BV families expansions
 


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Fig. 5. CDR3 size distribution of BV5 transcripts from PBMC, Miv (Lu 7.1) and LA (Lu 7.2) T cell lines of patient WFA. ß chain CDR3 amino acid sequences of the BV5–BJ2S3 oligoclonal peaks in Lu 7.1 and Lu 7.2 lines are indicated below the profiles. Percentages of BV5 family usage in comparison with all BV families or of the BJ segment expressed within BV5 are shown upper right to the profiles.

 
Therefore we sequenced the ß chain CDR3 regions from BV5–BJ2S3 expansions in the lines Lu 7.1 and Lu 7.2 from this patient in order to point out identical clones. Actually, among clones from Miv and LA HIL, six of six clones from Lu 7.1 had the same dominant sequence (SFDGSRTDTQ) and eight of nine clones from Lu 7.2 presented a difference at three amino acid residues (SFSGGFTDTQ) (Fig. 5Go). Only one clone from Lu 7.2 was identical with the dominant one found in Lu 7.1.

PBMC and intralesional T cell lines reactivity against streptococcal M5 peptides and heart tissue proteins
We studied the reactivity of PBMC and HIL from patients SLA, JSS, WFA and JEB against immunodominant N-terminal M5 peptides comprised of 1–25, 81–103 and 163–177 amino acids as previously described (33). The reactivity against several heart tissue protein fractions was also evaluated. Peptides comprised in regions 1–25 and 163–177 were recognized by PBMC of one patient, while peptides included in region 81–103 by three out of four patients. HIL from all patients recognized M5 peptides against regions 1–25 and 81–103. Streptococcal M5 (81–103) region contains an immunodominant peptide (81–96) more frequently recognized by both HIL and PBMC. Several heart tissue protein fractions were recognized by HIL from all patients, while PBMC from only two patients (SLA and JSS) recognized heart proteins (Table 4Go).


    Discussion
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 Methods
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 Discussion
 References
 
In post-streptococcal RHD the pathogenic role of infiltrating T cells, especially of the CD4 phenotype, is sustained by histopathological findings (32) and suggests that RHD might be an autoimmune disease. These cells could be triggered in several ways. An antigen-driven in situ immune response is supported by functional data showing the recognition of streptococcal M protein peptides and heart protein fractions (33). On the other hand, the possible superantigenic effect of Streptococcus pyogenes M protein (4752) was dismissed by Degnan and Fleischer (25,26) in studies utilizing recombinant M protein. Thus, these results assigned all superantigenic effects in streptococcal preparations to the pyrogenic exotoxins liberated by streptococci, that could act as an additional factor enhancing non-specifically the initial immune response. Differences in the recognition mechanism of superantigens and nominal antigens are reflected at the T cell repertoire level by respectively a polyclonal amplification of BV families or the expansion of discrete antigen-specific lymphocyte populations. The current analysis combining a semiquantitative estimate of BV gene family usage and a measurement of the ß chain CDR3 size diversity provides a powerful tool to distinguish between these two aspects of the immune response.

The possible effect of superantigens in human autoimmune diseases has not been as well established as in murine models, where a preferential usage of TCR V genes of autoreactive T cells has often been found (see review 24,53) with different patterns in different inbred mouse strains (54). In human diseases, for instance, an expansion of BV7 islet-infiltrating T cells in two insulin-dependent mellitus patients with extensive junctional diversity was suggestive of a superantigen effect (55). In PBMC of myasthenia gravis patients, it was found that BV families 1, 13.2, 17 and 20 were over-represented in both CD4+ and CD8+ T cells, suggesting a possible role of superantigen effect in this pathology (56). The results reported here in RHD patients are not consistent with a superantigenic effect of streptococcal infection on PBMC and HIL (Figs 1 and 2GoGo) of chronic RHD patients (JSS, JEB and NCM) or even in RHD patients who have had recrudescence of streptococcal infections in the previous last 6 months (patients ESS, WFA and SLA). Since the superantigenic streptococcal pyrogenic exotoxins (33) are liberated at the beginning of the streptococcal throat infection we cannot exclude, however, that certain TCR BV families may be preferentially expanded. However, our inability to find selective TCR BV family expansion in HIL from chronic RHD patients may indicate that this possible superantigenic effect during acute infection may not be relevant to heart damage.

Conversely, we found frequent oligoclonal expansions in heart-derived T cell lines from the different RHD patients (Figs 3 and 4GoGo). The possibility of TCR repertoire skewing due to in vitro culture is an important point which has been studied using the same technical procedures in tumor-infiltrating lymphocytes (TIL). A significant repertoire selection occurred in vitro only when TIL were expanded in the presence of the autologous tumor as antigen-presenting cells, and not when TIL were cultured in IL-2- and PHA-conditioned medium without stimulator cells or in the presence of autologous normal cells (57). Currier et al. (1996) performed Spectratyping on fresh PBMC and paired T cell lines stimulated with PHA for three cycles, and observed that TCR BV usage and CDR3 length diversity closely resembled those observed for unstimulated PBMC (31). We also observed that the BV frequency usage and CDR3 size diversity pattern of synovial fluid lymphocytes were not modified after two cycles of expansion on IL-2 and feeder cells as reported in this study (Dulphy and Toubert, unpublished observations). Therefore our in vitro culture conditions are unlikely to account for the HIL expansions. Although it has been reported that the same TCR {alpha}ß dimer could recognize different MHC–peptide complexes and vice versa (58), there is a large amount of evidence for the restriction of TCR gene usage after an antigenic challenge (30,31). Occurring even in the absence of an increase in the BV expression level, these major oligoclonal expansions found in HIL strongly support an antigen-specific T cell recognition and the in situ amplification of these lymphocyte populations. No common T cell expansion was observed between the different patients, probably because of their clinical heterogeneity. These results are in agreement with those reported in other human autoimmune diseases like reactive arthritis, insulin-dependent diabetes mellitus, multiple sclerosis or rheumatoid arthritis (46,55,59,60). There is a growing evidence for in situ antigen-driven responses in these diseases. For instance, in patients with multiple sclerosis some conserved CDR3 sequences in the BV5.2 family were found at the site of the inflammation in the brain, indicating a possible association of this shared hypervariable sequence with the development of the disease (59). Studies performed in patients with reactive arthritis, another bacterial-triggered autoimmune disorder, also showed multiple clonal T cell expansions in synovial infiltrates. The ß chain CDR3 motifs common to different patients have been defined but complete sequence identities between individuals were found only in case of a perfect HLA typing match and of recent-onset diseases (46).

The most noticeable result in this study was that surprisingly few oligoclonal BV expansions were shared by HIL from Miv and LA in a same individual (Fig. 3Go). This is in contrast with observations made in autoimmune diseases showing shared expansions at different pathological sites. Of note, these observations have been made mainly in rheumatic diseases (46,60) in which the joint antigen could be expected to be the same in different joints while in RHD the injured tissue could be more heterogeneous. The most straightforward example of our findings is the case of patient WFA with an expansion in the BV5 family at a 10 amino acid CDR3 size shared between Miv and LA intralesional T cells (Fig. 5Go). The thorough analysis of this expansion in both tissues showed that the same BJ2S3 segment was used, but that dominant amino acid CDR3 sequences were different at several amino acid residues indicating the presence of different dominant T cell clones in LA and in valvular tissue (Fig. 5Go). Taken together, the results suggest that the antigens recognized by infiltrating T cells in the LA and mitral tissue may not be the same and are in favor of a localized T cell expansion in the heart lesions, driven by specific antigenic peptides different at these two pathological sites consistent with muscle and connective tissue-derived proteins.

HIL of RHD patients studied here were predominantly CD4+ cells (Table 2Go), and all HIL showed reactivity against myocardium and valve tissue protein fractions. In addition, reactivity against the three immunodominant regions of streptococcal M5 protein, comprised between residues 1–25, 81–103 and 163–177 was also present (Table 4Go), further supporting the heart protein cross-reactive recognition of the same M5 epitopes by heart-infiltrating T cell clones (33). The streptococcal M5 (81–103) region comprises the immunodominant peptide M5 (81–96), which is frequently recognized by peripheral T lymphocytes, mainly from HLA-DR7/DR53+ severe RHD patients (Guilherme et al., submitted), suggesting a preferential presentation of this peptide to the TCR in the context of HLA DR7/DR53 molecules. Interestingly, it has been shown that mice immunized with human cardiac myosin display strong T cell proliferative responses that share sequences with M5 regions 81–103 (61), further supporting both the immunodominance and cross-reactivity of these regions. It is also known that M5 region 81–103 presents cross-reactivity with sarcolemma and cardiac myosin at the antibody level in mice, rabbits and humans (62,63,64). Indeed, Cunningham (65) suggested that the cross-reactivity may be due to the similar secondary structure found in cardiac myosin and M protein.

The most plausible explanation of the different T cell expansions at both heart sites is that after a common antigenic challenge towards the dominant M5 (81–96) peptide in the throat and following migration to different heart sites, a different spreading of the immune response could be triggered by different cross-reactive heart antigens. The fact that the HIL could react against multiple heart antigens (Table 4Go) is in agreement with this hypothesis and will deserve further biochemical purification of these antigens for a proper identification.

A major health problem in developing countries for RHD is severe patients with advanced cardiac injuries who can only benefit from surgery. Large-scale educational programs and antibiotic treatment of streptococcal infections are undoubtedly most important actions (66) but are a difficult goal to achieve and will not solve the problem of established RHD. The research of auto-antigens implicated with the progression of heart lesions could help us to understand the mechanism underlying autoimmunity in this pathology and also result in a new way of therapy attempting to prevent the generation of definitive heart injuries.


    Acknowledgments
 
We thank Drs P. Kourilsky, C. Pannetier and J. Even for providing us with the Immunoscope software. The technical expertise of Mrs Sandra M. Monteiro on flow cytometry experiments is acknowledged. This work was supported by grants from the PADCT-CNPq no. 620087/94.3 and HHMI 75197-555101, and USP-Cofecub 25-96. N. D. is a recipient of a grant from the Fondation pour la Recherche Médicale (FRM) and Association de la Recherche sur la Polyarthrite (ARP).


    Abbreviations
 
CDR complementary-determining region
HIL heart infiltrating T cell lines
LA left atrium
Miv mitral valve
PBMC peripheral blood mononuclear cell
PHA phytohemagglutinin
RF rheumatic fever
RHD rheumatic heart disease
TIL tumor-infiltrating lymphocyte

    Notes
 
DC, AT and JK should be considered as senior co-authors.

Transmitting editor: J.-F. Bach

Received 17 November 1999, accepted 23 March 2000.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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