International Immunology, Vol. 12, No. 7, pp. 1063–1074 © 2000 The Japanese Society for Immunology

Molecular evidence for antigen-drivenimmune responses in cardiac lesions ofrheumatic heart disease patients

Luiza Guilherme1, Nicolas Dulphy5, Corinne Douay5, Veronica Coelho1,Edecio Cunha-Neto1, Sandra E. Oshiro1, Raimunda V Assis1,4, Ana C. Tanaka1,Pablo M. Alberto Pomerantzeff 1, Dominique Charron5, Antoine Toubert5 andJorge Kalil1,2,3

1Heart Institute–InCor, University of Sao Paulo, School of Medicine, and 2Clinical Immunology and Allergy,Department of Clinical Medicine, and 3International Scholar Howard Hughes Medical Institute, Sao Paulo,Brazil4University of Juiz de Fora, Department of Pathology, Minas Gerais, Brazil5Laboratoire d’Immunologie et d’Histocompatibilite, INSERM U396, Institut Universitaire d’Hematologie,Hopital St Louis, Paris, France

Key words: autoimmunity, M protein, rheumatic heart disease, superantigens, TCR

Abstract

Rheumatic heart disease (RHD) is a sequel of post-streptococcal throat infection. Molecularmimicry between streptococcal and heart components has been proposed as the triggering factorof the disease, and CD4� T cells have been found predominantly at pathological sites in the heartof 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 workwe focused on the TCR β chain family (TCR BV) usage and the degree of clonality assessed by βchain complementarity-determining region (CDR)-3 length analysis. We have shown that in chronicRHD 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 cellexpansions were more frequently observed in HIL than in PBMC. Some major BV expansions wereshared between the mitral valve (Miv) and left atrium (LA) T cell lines, but an in-depth analysis ofBJ segments usage in these shared expansions as well as nucleotide sequencing of the CDR3regions suggested that different antigenic peptides could be predominantly recognized in the Mivand the myocardium. Since different antigenic proteins probably are constitutively represented inmyocardium and valvular tissue, these findings could suggest a differential epitope recognition atthe two lesional heart sites after a common initial bacterial challenge.

Introduction

Rheumatic fever (RF) is a consequence of throat infection by Antigenic mimicry between streptococcal antigens, mainly Mprotein epitopes, and heart components has been proposedβ-hemolytic group A streptococci, affecting 3–4% of non-

treated children and therefore a major health problem in as the triggering factor leading to autoimmunity in individualswith genetic predisposition (1–3). Several genetic markers ofBrazil. Rheumatic heart disease (RHD) develops 4–8 weeks

or later after streptococcal infection in ~30% of individuals susceptibility were studied and no consistent association wasfound (4); however, associations with different HLA class IIwith RF.

The pathogenic mechanisms involved in the development antigens have been observed in several populations (5–13).Since HLA class II antigens play an important role in theof RF/RHD remain unclear; however, it is evident that an

abnormal humoral and cellular immune response occurs. antigen presentation to the TCR, the variable association with

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

Correspondence to: L. Guilherme, Laboratorio de Imunologia de Transplantes, Instituto do Coracao, HC-FMUSP, Av. Dr Eneas de CarvalhoAguiar, 500–3° andar 05403-000, Sao Paulo, SP, Brazil

Transmitting editor: J.-F. Bach Received 17 November 1999, accepted 23 March 2000

1064 Oligoclonal T cell expansions in rheumatic heart disease lesions

HLA antigens is consistent with the possibility that different and the amino acid sequences of selected T cell expansionsshared at different sites in the heart.serotypes of group A streptococci could be implicated in the

disease in different countries. M proteins are the major targetsof the host anti-streptococcal immune response (14). The

Methodsheart is considered as an immunocompetent organ withlymph node drainage, and is consequently under immune Patient samplessurveillance by lymphocytes and macrophages. Dendritic

Peripheral blood mononuclear cells (PBMC) were obtainedcells expressing HLA class I and class II molecules at theirfrom six patients with severe RHD, from the Heart Institutesurface and able to present antigens to T lymphocytes haveHC–FMUSP, selected according to Jones’ modified criteriabeen described in the heart (15). In acute RF, Aschoff bodies(34). The average length of patient follow-up was 5 years.(conglomerates of monocytes/macrophages and neutrophils)Blood samples were taken in the absence of immunosuppres-are frequently found and play an important role in thesive drugs. Patient data and samples used in TCR studiestriggering of a local inflammatory process, acting as antigen-are summarized in Table 1. Both blood samples and surgicalpresenting cells (16,17). Autoreactivity to heart antigensfragment collection procedures were cleared by the Commit-caused by microbial infections was described in several hearttee of Ethics of the Heart Institute, HC–FMUSP.

diseases (18–20). Superantigens are proteins derived frombacteria and viruses that polyclonally activate T cells by an PBMC and T cell linesMHC class-II dependent, but haplotype-unrestricted, mech-

PBMC were obtained by density gradient centrifugationanism (21–23). Proliferative responses to superantigens are(Ficoll-Hypaque) and were isolated directly by three success-

limited to T cells expressing particular TCR BV gene familiesive washes in PBS. Heart-infiltrating T cell lines (HIL) were

and are independent of antigen specificity. Superantigens derived from surgical heart fragments obtained during valveare known to activate autoreactive T cells and initiate an correction in four of these patients. Two patients (WFA andinflammatory process leading to autoimmunity in animal JSS) yielded two T cell lines, derived from mitral valve (Miv)models, although a definite role for superantigens in the and left atrium (LA). For patients SLA and JEB T cell linespathogenesis of human autoimmune diseases is still a matter were derived from Miv and LA respectively. Tissue was finelyof debate (24). Several streptococcal pyrogenic exotoxins, minced with injection needles and small scissors, placed inbut not M5 protein, display superantigenic properties (25–27). Falcon flat-bottom 96 multi-well plates (Becton Dickinson,

The TCR β chain is produced by the assembly of variable Lincoln Park, NJ), with DMEM (Sigma, St Louis, MO) supple-(V)–diversity (D)–joining (J) and constant (C) gene segments. mented with 2 mM L-glutamine (Sigma), 10% pooled nor-The V–D–J junction encodes the hypervariable region of mal human serum, 10 mM HEPES (Sigma), antibioticsthe TCR designated as complementarity-determining region (Gentamycin and Peflacyn) at the concentration of 40 and 20(CDR)-3 that interacts directly with the complex peptide–MHC µg/ml respectively, and 40 U/ml of human recombinant IL-2molecule. Two methods called Spectratyping and Immunos- (Biosource, Camarillo, CA), on a HLA-DR matched feedercopy have been described (28,29) in order to determine the layer of PBMC at 105 cells/well, irradiated at 5000 rad (35,36).size of CDR3 regions in transcripts of whole BV families or in

HLA typingBV–BJ combinations, and are particularly suitable for analysisof αβ T cell clonality in tissue-infiltrating T cells (30). Patterns Patients were typed for HLA-DR and -DQ by PCR reactions.of β chain CDR3 diversity are definitely different in the case Briefly, 24 PCR reactions were performed per patient, 20 forof activation by superantigens or nominal antigens giving rise assigning HLA-DR1 to -DR18, one each for HLA-DR51, -DR52respectively to polyclonal expansions of whole BV families or and -DR53, and one amplification control. For DQ1 to DQ9to the disturbance of the Gaussian distribution of CDR3 size specificities, eight different PCR reactions were sufficient (37).by one or several oligoclonal expansions (31).

ImmunohistochemistryThe presence of CD4� T cells in the heart of RHD patientshas been demonstrated, suggesting a direct role for these Sections of 4 µm were cut from cardiac tissue prepared fromcells in the pathogenesis of RHD (32). In a previous work, we frozen surgical fragments and specimens embedded in OCTshowed that T lymphocyte clones infiltrating the heart lesions 4583 (Miles, Naperville, IL). Anti-CD4 (MT 310) and CD8 (DKof severe RHD patients are able to recognize myocardium 25) (Dakopatts, Glostrup, Denmark) mAb were used to defineand valve proteins as well as immunodominant streptococcal T cell subpopulations. Peroxidase-coupled avidin (Dakopatts)M5 peptides, a finding suggestive of molecular mimicry at was added later and the reaction was developed with diamino-the T cell level between β hemolytic streptococci and heart benzidine (Sigma).tissue (33). The aim of the present work was to compare the

FACSTCR BV usage in peripheral blood and heart-infiltrating T celllines (HIL) from RHD patients, looking for oligoclonal β chain T cell subpopulations from intralesional T cell lines wereexpansions in line with antigen-driven immune responses, analyzed using anti-αβ TCR–FITC (T10B9.1A-31) (PharM-and for a possible superantigenic effect still detectable at ingen, San Diego, CA), anti-CD3–FITC (UCTH1), CD4–phyco-chronic RHD patients. We also wanted to evaluate the com- erythrin (MT310) and CD8–FITC (DK 25) mAb (Dakopatts). Inplexity of this response at different pathological sites in the total, 5�103 events gated on lymphocytes were analyzedsame and in different patients by determining the β chain using a FACScan flow cytometer with CellQuest software

(Becton Dickinson, Mountain View, CA).CDR3 size profile, and by defining the BJ segment usage

Oligoclonal T cell expansions in rheumatic heart disease lesions 1065

Table 1. Identification and HLA-DR/DQ of patients, clinical and histopathology characteristics and samples analysed forTCR studies

Patients Gender Age HLA-DR/DQ Clinical date Histopathology Samples analyzed

SLA M 6 15, 7, 51, 53/6, 2 Severe Miv regurgitation Chronic valvulitis, Aschoff PBMC, T Line Lu 3.1 (Miv)and moderate aortic valve nodules in papillar muscleregurgitation

WFA M 15 17, 13, 52/ 6, 2 Severe mitral and aortic Acute mitral valvulitis; PBMC, T Line Lu 7.1valve regurgitation myocardium fibrosis and fibers (Miv), T Line Lu 7.2 (LA)

hypertrophyJSS M 10 9, 11, 52, 53/2, 7 Severe mitral and aortic Chronic rheumatic valvulitis PBMC, T Line Lu 4.1

valves regurgitation Myocardium lymphohistiocytary (Miv), T Line Lu 4.2 (LA)reactivity, fibrosis and fibershypertrophy

JEB M 15 15, 7, 51, 53/ 6, 2 Severe Miv regurgitation Mitral valve interstitial fibrosis PBMC, T Line Lu 5 (LA)and fibers hypertrophy

NCM M 16 11, 13, 52/ 5, 7 Severe Miv regurgitation Myocardium fibers hypertrophy PBMCESS F 13 4, 7, 53/6, 2 Severe Miv regurgitation Acute and chronic mitral PBMC

and moderate aortic valve valvulitisregurgitation

RNA extraction and cDNA synthesis in the PCR fluorescent BV–BC products depends only on thesize of the V–D–J junctions. Statistical analysis was performedRNA was extracted from cell pellets of intralesional T cellto determine whether or not a profile could be considered aslines (8–10 days after the first or second phytohemagglutininGaussian: a profile was not considered to be Gaussian if onestimulation in the presence of IL-2 and feeder cells) andpeak was excluded from the 95% confidence interval of peakPBMC by lysis in guanidium thiocyanate buffer. RNA extractedlevel intensities. TCR B subfamilies BV10 and BV19 werefrom paired wells containing 5�106 irradiated feeder cells,omitted from this analysis as they are pseudogenes in mostPHA, IL-2 and incubated for the same 8–10 days was unde-individuals (40).tectable. The PBMC and HIL cDNA were prepared from 1–

10 µg total RNA with AMV reverse transcriptase (cDNA cycle BV and BJ gene usagekit; Invitrogen, Leek, The Netherlands) as described (38).

PBMC cDNA samples were quantified as described (38), and~3�106 copies of cDNA from each PBMC sample were thenOligonucleotides and CDR3 size analysisamplified for 30 cycles with the BV primers and an internal

The nomenclature of BV families and the primers used havefluorescent BC primer. BJ usage was defined only for some

been described (38,39). Fluorescent primers for BC and BJintralesional T cells expressing oligoclonal BV families after

were labeled at the 5� end with the Fam fluorophore (Appliedrun-off reactions of the unlabeled BV–BC amplification product

Biosystems, Foster City, CA). Aliquots of the cDNA synthesisand is quantitative since the fluorescent primers have compar-

reaction (corresponding to 250 ng of total RNA) were amplifiedable amplification efficiencies (30). The fluorescence intensity

in 50 µl reactions with one of the BV-specific oligonucleotidesin each BV or BJ family was expressed as the percentage of

as the 5� primer and the BC oligonucleotide as the 3� primer.total signals from the 22 BV or 13 BJ subfamilies.

The final concentration was 0.5 mM for each primer, dNTP0.2 mM, MgCl2 2 mM in Taq polymerase buffer (Promega, DNA sequencingMadison, WI) in the presence of 1 U of Taq polymerase BVBJ PCR products were cloned into pCR®2.1 vector (Invi-(Promega) on a DNA thermal cycler (9600; Perkin-Elmer, trogen) and transformed into XL1 Blue supercompetent cellsNorwalk, CT). The PCR cycle profile was : denaturation at (Stratagene, La Jolla, CA). After blue/white screening of94°C for 30 s, annealing at 60°C for 45 s, primer extension recombinant plasmids on X-galactoside/isopropylthiogalacto-at 72°C for 45 s for 40 cycles and a final polymerization step side (IPTG) indicator plates, plasmids were purified by alkalineof 5 min at 72°C. Aliquots from each BV–BC PCR product (2 lysis followed by phenol–chloroform–iso-amyl alcohol. Insertsµl) were copied in six-cycle run-off reactions primed with were checked by agarose gel electrophoresis after BV–BJa fluorophore-labeled BC or BJ oligonucleotide. The final PCR amplification and both strands were sequenced with theconcentration of dNTP was 0.2 mM, 3 mM MgCl2 in the ABI Prism Dye Primer Cycle sequencing kit (Perkin-Elmer).presence of 0.2 U of Taq polymerase. The run-off reactions Products were loaded on 4.25% acrylamide sequencing gelswere migrated on 4.25% acrylamide sequencing gels (377A (377A DNA sequencer; Applied Biosystems) and analyzedDNA sequencer; Applied Biosystems) for size (Genescan- with the Sequence Navigator software.500 size marker; Perkin-Elmer) and fluorescence intensity

Peptide synthesis and preparation of human heart tissuedetermination. The raw data were analyzed with the help of theprotein fractionsImmunoscope software (29). The CDR3 region was defined to

include residues 95–106 (30). Since the positions of the BV Peptides based on the published M5 protein sequence (41,42)were synthesized by the ‘tea bag’ method by t-BOC chemistryand the BC primers are fixed, the length distribution observed

1066 Oligoclonal T cell expansions in rheumatic heart disease lesions

Table 2. Surface phenotype of T cells from surgical samples and T cell lines derived in vitro

Patient Tissue samples (positive cells/field) T cell Lines (% positive by flow cytometry)

Tissue CD4 CD8 CD4/CD8 T cell line identification αβ TCR CD3 CD4 CD8 CD4/CD8

SLA Miv 3.2 2.4 1.3 Lu 3.1 94.4 99.8 87.2 7.4 11.8WFA Mi v 1.2 1.2 1.0 Lu 7.1 94.2 99.1 49.4 38.5 1.3

LA 4.5 0.9 5.0 Lu 7.2 67.5 99.2 22.0 29.0 0.8JSS Miv NT NT NT Lu 4.1 95.8 97.7 91.0 6.0 15.2

LA 6.1 1.2 5.1 Lu 4.2 93.0 99.9 76.6 8.5 9.0JEB LA NT NT NT Lu 5 NT 98.4 86,0 0.1 860

NT, not tested.

(43), and were checked by mass spectrometry and HPLC. Level of expression of TCR BV gene in PBMC and intralesionalT cell linesTissue fractions from human ventricular myocardium, aortic

valve, were obtained from lysates of post-mortem normal We measured by a semiquantitative analysis the frequencytissue samples, separated by SDS–PAGE and blotted onto of 22 TCR BV families in PBMC from six severe RHD patientsnitrocellulose membranes (Sigma) (44,45). The blots were and the intralesional T cell lines described in Tables 1 and 2.divided in several horizontal strips with approximately the The relative frequency in both periphery and intralesional Tsame amount of protein with defined mol. wt values as cell cultures showed no particular expansion of any BV family.previously described (33). The strips were solubilized in Some BV families (BV2, BV3, BV6 and BV22) (Fig. 1) wereDMSO (Merck, Darmstadt, Germany), precipitated in sodium highly expressed in the periphery (�8.0% each) for most ofcarbonate/sodium bicarbonate buffer 0.05 M, pH 9.6, and the patients. In intralesional T cell cultures the most frequentwashed with RPMI 1640 medium (Sigma, St Louis, MO) to BV families were BV2, BV3, BV6 and BV22 (Fig. 2). The overallyield a fine suspension of protein-loaded nitrocellulose (see pattern of BV gene expression was similar in PBMC versusTable 4 for peptides sequences and heart proteins mol. wt). HIL, but when compared in single individuals some BV families

appeared to be more expressed among PBMC than in HILProliferation assaysor vice versa. For instance, in patient WFA, BV6 was more

Proliferation assays were performed in Falcon flat-bottom 96 expressed in the periphery than in Miv HIL (15.0 versusmultiwell plates (Becton Dickinson, Lincoln Park, NJ), using 5.7% respectively) while BV15 was more highly expressed in105 mononuclear cells/well isolated from peripheral blood by myocardium-derived T cells (8.0 versus 1.0 and 1.5% in Miv-centrifugation on a d � 1077 density gradient, with 5 µg/ml derived T cells and in the periphery respectively). There wasof streptococcal M5 peptides or 20 µl/well of heart tissue no statistical difference in PBMC BV usage in RHD patientsfractions added for 96 h at 37°C in a humidified 5% CO2 compared to normal individuals tested under identical tech-incubator. Negative controls were mononuclear cells in DMEM nical procedures (38,46).(Sigma) without antigens for the peptide experiments and 20µl of a protein-free nitrocellulose suspension for heart-tissue CDR3 size patterns in TCR BV familiesfraction experiments. PHA-P (5 µg/ml) (Sigma) was used for

The analysis of TCR BV and BJ genes using the Immunoscopepositive control of proliferative responses. Triplicate wells wereapproach allows an evaluation of the T cell diversity inpulse-labeled with 1 µCi/well of [3H]thymidine (Amersham Lifegiven BV families by analyzing CDR3 size patterns (29). TheSciences, London, UK) for the final 18 h of culture; cells wereobtained BV–BC patterns were grouped according to theirharvested and analysed in a automated gas-phase β-counterpatterns into three groups as: oligoclonal profiles, one or two(Matrix 96; Packard, Camberra, Australia). For T cell lines wepeaks above the Gaussian background and Gaussian profilesused 2�104 T cells/well with 105 HLA-DR-matched irradiated(Fig. 3). Our results showed that oligoclonal T cell expansionsPBMC (5000 rad) for 72 h. The proliferative response wasare found more frequently in HIL than in PBMC. Few oligo-considered positive when the Stimulation Index (SI � meanclonal expansions were found in the PBMC (BV8 and BV23experimental c.p.m./negative control c.p.m.) was �2.5.in SLA; BV7 and BV8 in JEB; BV18 in JSS; BV4, BV8 andBV15 in NCM and BV8 in ESS). Patient WFA presented notrue oligoclonal expansion in PBMC with one or at most twoResultspeaks above the Gaussian background (Fig. 3).

T cell subsets in heart tissue and intralesional T cell lines The CDR3 size distribution of major TCR BV expansions ofPBMC and T cell lines of patients JEB, JSS, SLA and WFAImmunohistochemical determination was performed on heart

tissue fragments of three patients (SLA, WFA and JSS) and are presented in Fig. 4. When we compared the patterns ofthe CDR3 size diversity in PBMC and intralesional T cellCD4� T cells were predominantly found (Table 2). Six intra-

lesional T cell lines were generated from Miv and LA (myocar- cultures we found several pictures: polyclonal families in bothPBMC and intralesional T cell cultures (e.g. BV6 in patientdium) fragments. All T cell lines were predominantly CD3�

and αβ TCR� and most (five of six) were also predominantly JEB); oligoclonal BV expansions at one site only (BV22 inJSS Lu 4.2 and BV24 in JSS Lu 4.1; BV4 in WFA Lu 7.1 andCD4� (Table 2).

Oligoclonal T cell expansions in rheumatic heart disease lesions 1067

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

BV13 in WFA Lu 7.2); oligoclonal expansions in PBMC and three amino acid residues (SFSGGFTDTQ) (Fig. 5). Only oneclone from Lu 7.2 was identical with the dominant one foundheart 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 in Lu 7.1.we found a polyclonal profile in PBMC and oligoclonal peaks

PBMC and intralesional T cell lines reactivity against strepto-in both LA and Miv T cell lines at a different (BV13 in WFA Lucoccal M5 peptides and heart tissue proteins7.1 and Lu 7.2) or at the same CDR3 size (BV1 and BV23 in

JSS; BV5 in WFA). We studied the reactivity of PBMC and HIL from patients SLA,JSS, WFA and JEB against immunodominant N-terminal M5

TCR BJ usage in expanded TCR BV families peptides comprised of 1–25, 81–103 and 163–177 aminoacids as previously described (33). The reactivity againstThe finding of oligoclonal expansions with the same CDR3

length in some BV families led us to look at the dominant BJ several heart tissue protein fractions was also evaluated.Peptides comprised in regions 1–25 and 163–177 wereusage in major TCR BV expansions of HIL from JSS, WFA,

SLA and JEB patients (Table 3). In some BV expansions we recognized by PBMC of one patient, while peptides includedin region 81–103 by three out of four patients. HIL from allfound a unique BJ segment, e.g. BJ2S3 in the BV1 expansion

for patient SLA or BV17–BJ1S3 for patient JEB. In other patients recognized M5 peptides against regions 1–25 and81–103. Streptococcal M5 (81–103) region contains an immu-cases several BJ segments accounted for the BV–BC major

expansion as for instance in the BV23 expansions (10 amino nodominant peptide (81–96) more frequently recognized byboth HIL and PBMC. Several heart tissue protein fractionsacids) found in common in Miv and LA HIL of patient JSS.

Notably, the multiple BJ segments were different in the two were recognized by HIL from all patients, while PBMC fromonly two patients (SLA and JSS) recognized heart proteinsHIL from this patient. The patient WFA had also an oligoclonal

BV5 expansion at a 10 amino acids CDR3 size in both HIL (Table 4).Lu 7.1 (Miv) and Lu 7.2 (LA). Conversely, in this case thesame BJ2S3 gene segment was used in both intralesional

DiscussionT cell lines (Table 3 and Fig. 5).Therefore we sequenced the β chain CDR3 regions from In post-streptococcal RHD the pathogenic role of infiltrating

T cells, especially of the CD4 phenotype, is sustained byBV5–BJ2S3 expansions in the lines Lu 7.1 and Lu 7.2 fromthis patient in order to point out identical clones. Actually, histopathological findings (32) and suggests that RHD might

be an autoimmune disease. These cells could be triggeredamong clones from Miv and LA HIL, six of six clones from Lu7.1 had the same dominant sequence (SFDGSRTDTQ) and in several ways. An antigen-driven in situ immune response

is supported by functional data showing the recognition ofeight of nine clones from Lu 7.2 presented a difference at

1068 Oligoclonal T cell expansions in rheumatic heart disease lesions

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

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

Oligoclonal T cell expansions in rheumatic heart disease lesions 1069

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); LAT cell lines (Lu 5, Lu 4.2 and Lu 7.2). The 10 amino acids CDR3 size is indicated on the abcisses scale.

streptococcal M protein peptides and heart protein fractions The possible effect of superantigens in human autoimmunediseases has not been as well established as in murine(33). On the other hand, the possible superantigenic effect

of Streptococcus pyogenes M protein (47–52) was dismissed models, where a preferential usage of TCR V genes ofautoreactive T cells has often been found (see review 24,53)by Degnan and Fleischer (25,26) in studies utilizing recombin-

ant M protein. Thus, these results assigned all superantigenic with different patterns in different inbred mouse strains (54).In human diseases, for instance, an expansion of BV7 islet-effects in streptococcal preparations to the pyrogenic exotox-

ins liberated by streptococci, that could act as an additional infiltrating T cells in two insulin-dependent mellitus patientswith extensive junctional diversity was suggestive of a super-factor enhancing non-specifically the initial immune response.

Differences in the recognition mechanism of superantigens antigen effect (55). In PBMC of myasthenia gravis patients, itwas found that BV families 1, 13.2, 17 and 20 were over-and nominal antigens are reflected at the T cell repertoire

level by respectively a polyclonal amplification of BV families represented in both CD4� and CD8� T cells, suggesting apossible role of superantigen effect in this pathology (56).or the expansion of discrete antigen-specific lymphocyte

populations. The current analysis combining a semiquantit- The results reported here in RHD patients are not consistentwith a superantigenic effect of streptococcal infection onative estimate of BV gene family usage and a measurement

of the β chain CDR3 size diversity provides a powerful tool PBMC and HIL (Figs 1 and 2) of chronic RHD patients (JSS,JEB and NCM) or even in RHD patients who have hadto distinguish between these two aspects of the immune

response. recrudescence of streptococcal infections in the previous

1070 Oligoclonal T cell expansions in rheumatic heart disease lesions

Table 3. BJ gene segments in relevant BV families expansions

Sample BV Family Relative percent BJ Segments Relative percent CDR3 size

SLA Lu3.1 1 2.5 2.3 100.00 118 4.1 1.1 42.6 9

2.7 16.7 813 8.3 1.5 11.1 10

2.7 61.8 1023 10.5 2.2 24.8 10

2.3 18.8 11

JSS Lu 4.1 1 7.6 1.2 17.5 81.6 40.3 102.5 11.1 10

2 6.9 2.1 24.5 102.7 48.0 10

23 5.5 1.2 28.1 81.5 11.4 101.6 20.5 102.5 13.1 10

JSS Lu 4.2 1 7.1 1.1 45.8 102.1 12.0 10

23 2.7 1.1 47.6 102.1 13.0 102.3 16.6 10

10

WFA Lu 7.1 5 5.4 2.3 24.0 102.7 53.0 10

WFA Lu 7.2 5 8.6 2.3 32.0 10

JEB Lu 5 17 15.8 1.3 94.4 11

Mitral valve T cell lines: Lu 3.1, Lu 4.1 and Lu 7.1. Left atrium T cell lines: Lu 4.2, Lu 7.2 and Lu 5. Underlined, oligoclonal expansionsshared by mitral and LA T cell lines. Only oligoclonal BJ gene segments with �10% of expression were presented.

last 6 months (patients ESS, WFA and SLA). Since the and Toubert, unpublished observations). Therefore our invitro culture conditions are unlikely to account for the HILsuperantigenic streptococcal pyrogenic exotoxins (33) are

liberated at the beginning of the streptococcal throat infection expansions. Although it has been reported that the same TCRαβ dimer could recognize different MHC–peptide complexeswe cannot exclude, however, that certain TCR BV families

may be preferentially expanded. However, our inability to find and vice versa (58), there is a large amount of evidence forthe restriction of TCR gene usage after an antigenic challengeselective TCR BV family expansion in HIL from chronic RHD

patients may indicate that this possible superantigenic effect (30,31). Occurring even in the absence of an increase in theBV expression level, these major oligoclonal expansions foundduring acute infection may not be relevant to heart damage.

Conversely, we found frequent oligoclonal expansions in in HIL strongly support an antigen-specific T cell recognitionand the in situ amplification of these lymphocyte populations.heart-derived T cell lines from the different RHD patients (Figs

3 and 4). The possibility of TCR repertoire skewing due to No common T cell expansion was observed between thedifferent patients, probably because of their clinical hetero-in vitro culture is an important point which has been studied

using the same technical procedures in tumor-infiltrating geneity. These results are in agreement with those reportedin other human autoimmune diseases like reactive arthritis,lymphocytes (TIL). A significant repertoire selection occurred

in vitro only when TIL were expanded in the presence of the insulin-dependent diabetes mellitus, multiple sclerosis orrheumatoid arthritis (46,55,59,60). There is a growing evid-autologous tumor as antigen-presenting cells, and not when

TIL were cultured in IL-2- and PHA-conditioned medium ence for in situ antigen-driven responses in these diseases.For instance, in patients with multiple sclerosis some con-without stimulator cells or in the presence of autologous normal

cells (57). Currier et al. (1996) performed Spectratyping on served CDR3 sequences in the BV5.2 family were found atthe site of the inflammation in the brain, indicating a possiblefresh PBMC and paired T cell lines stimulated with PHA for

three cycles, and observed that TCR BV usage and CDR3 association of this shared hypervariable sequence with thedevelopment of the disease (59). Studies performed inlength diversity closely resembled those observed for unstimu-

lated PBMC (31). We also observed that the BV frequency patients with reactive arthritis, another bacterial-triggeredautoimmune disorder, also showed multiple clonal T cellusage and CDR3 size diversity pattern of synovial fluid

lymphocytes were not modified after two cycles of expansion expansions in synovial infiltrates. The β chain CDR3 motifscommon to different patients have been defined but completeon IL-2 and feeder cells as reported in this study (Dulphy

Oligoclonal T cell expansions in rheumatic heart disease lesions 1071

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. β chain CDR3 aminoacid sequences of the BV5–BJ2S3 oligoclonal peaks in Lu 7.1 and Lu 7.2 lines are indicated below the profiles. Percentages of BV5 familyusage in comparison with all BV families or of the BJ segment expressed within BV5 are shown upper right to the profiles.

Table 4. Reactivity of intralesional T cell lines and peripheral blood T cells from severe RHD patients against immunodominantstreptococcal M5 protein region and heart tissue protein fractions

Patients/HLA DR T cell lines Immunodominant Heart protein fractions Immunodominant Heart protein fractionsM5 region (mol. wt, kDa) M5 region (mol. wt, kDa)

SLA 15, 7,51,53 Lu 3.1 1–25, 81–103,163–177 Myoc- 24–30; 30–44 PBMC 1–25, 81–103, Myoc –12–24163–177

AoV –10–30 to 90–150WFA 17,13,52 Lu 7.1 1–25, 81–103, 163–177 Myoc 12–24; 44–65; �150 PBMC negative negative

AoV 30–43 to �150JSS9,11,52,53 Lu 4.2 1–25, 81–103, 163–177 Myoc 12–24 to �150 PBMC 81–96 Myoc �150

Ao 10–30 to �150 Aov – 30–43 �150Lu 4.1 1–25, 81–103 Ao 30–43 to 90–150

JEB15,7,51,53 Lu 5 11–25, 81–96 Myoc 90–150 PBMC 81–96 negativeAo V 30–43 to 65–90

Left atrium-derived T cell lines (Lu 4.2, Lu 7.2 and Lu 5). Mitral valve-derived T cell lines (Lu 3.1, Lu 4.1 and Lu 7.1). Aov, aortic valve-derived proteins, Myoc, myocardium-derived proteins. Positive reactions were considered when SI � 2.5; Lu 7.2, not tested. Peptidessequences 1–25, TRGTISDPQRAKEALDKYELENH; 81–103, DKLKQQRDTLSTQKETLEREVQN; 163–177, ETIGTLKKILDETVK (33).

sequence identities between individuals were found only in the same in different joints while in RHD the injured tissuecould be more heterogeneous. The most straightforwardcase of a perfect HLA typing match and of recent-onset

diseases (46). example of our findings is the case of patient WFA with anexpansion in the BV5 family at a 10 amino acid CDR3 sizeThe most noticeable result in this study was that surprisingly

few oligoclonal BV expansions were shared by HIL from Miv shared between Miv and LA intralesional T cells (Fig. 5). Thethorough analysis of this expansion in both tissues showedand LA in a same individual (Fig. 3). This is in contrast with

observations made in autoimmune diseases showing shared that the same BJ2S3 segment was used, but that dominantamino acid CDR3 sequences were different at several aminoexpansions at different pathological sites. Of note, these

observations have been made mainly in rheumatic diseases acid residues indicating the presence of different dominantT cell clones in LA and in valvular tissue (Fig. 5). Taken(46,60) in which the joint antigen could be expected to be

1072 Oligoclonal T cell expansions in rheumatic heart disease lesions

together, the results suggest that the antigens recognized by Abbreviationsinfiltrating T cells in the LA and mitral tissue may not be the CDR complementary-determining regionsame and are in favor of a localized T cell expansion in the HIL heart infiltrating T cell linesheart lesions, driven by specific antigenic peptides different LA left atrium

Miv mitral valveat these two pathological sites consistent with muscle andPBMC peripheral blood mononuclear cellconnective tissue-derived proteins.PHA phytohemagglutinin

HIL of RHD patients studied here were predominantly RF rheumatic feverCD4� cells (Table 2), and all HIL showed reactivity against RHD rheumatic heart disease

TIL tumor-infiltrating lymphocytemyocardium and valve tissue protein fractions. In addition,reactivity against the three immunodominant regions of strep-tococcal M5 protein, comprised between residues 1–25,

References81–103 and 163–177 was also present (Table 4), furthersupporting the heart protein cross-reactive recognition of the 1 Kaplan, M. H. and Suchy, M. L. 1964 Immunological relation ofsame M5 epitopes by heart-infiltrating T cell clones (33). The streptococcal and tissue antigens. II. Cross-reactions of antisera

to mammalian heart tissue with a cell wall constituent of certainstreptococcal M5 (81–103) region comprises the immunodom-strains of group A streptococci. J. Exp. Med. 119:643inant peptide M5 (81–96), which is frequently recognized

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genetics of rheumatic fever: relationship to streptococcal infectiondisplay strong T cell proliferative responses that share and autoimmune disease. J. Rheumatol. 18 (suppl 30):1.sequences with M5 regions 81–103 (61), further supporting 5 Ayoub, E. M., Barrett, D. J., MacLaren, N. K. and Krischen, J. P.

1986. Association of class II histocompatibility leukocyte antigensboth the immunodominance and cross-reactivity of thesewith rheumatic fever. J. Clin. Invest. 77:2019.regions. It is also known that M5 region 81–103 presents

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expansions at both heart sites is that after a common antigenic C. and Bhatia, M. L. 1986. HLA, blood groups and secretor statusin patients with established rheumatic fever and rheumatic heartchallenge towards the dominant M5 (81–96) peptide in thedisease. Tissue Antigens 27:172.throat and following migration to different heart sites, a different

9 Guilherme, L., Weidebach, W., Kiss, M. H., Snitcowsky, R. andspreading of the immune response could be triggered byKalil, J. 1991. Association of human leukocyte class II antigens

different cross-reactive heart antigens. The fact that the HIL with rheumatic fever or rheumatic heart disease in a Braziliancould react against multiple heart antigens (Table 4) is in population. Circulation 83:1995.

10 Weidebach, W., Goldberg, A. C., Chiarella, J., Guilherme, L.,agreement with this hypothesis and will deserve furtherSnitcowsky, R., Pileggi, F. and Kalil, J. 1994. HLA class II antigensbiochemical purification of these antigens for a proper identi-in rheumatic fever: analysis of the DR locus by RFLP andfication. oligotyping. Hum. Immunol. 40:253.

A major health problem in developing countries for RHD is 11 Maharaj, B., Hammond, M. G., Appadoo, B., Leary, W. P. andPudifin, D. J. 1987. HLA-A, B, DR and DQ antigens in blacksevere patients with advanced cardiac injuries who can onlypatients with severe chronic rheumatic heart disease.benefit from surgery. Large-scale educational programs andCirculation 76:259.antibiotic treatment of streptococcal infections are

12 Olmez, U., Turgay, M., Ozenirler, S., Tutkak, H., Duzgun, N.,undoubtedly most important actions (66) but are a difficult Duman, M. and Tokgoz, G. 1992. Association of HLA class I andgoal to achieve and will not solve the problem of established class II antigens with rheumatic fever in a Turkish population.

Rheumatology 22:49.RHD. The research of auto-antigens implicated with the13 Guedez, Y., Kotby, A., El-Demellawy, M., Galal, A., Thomson, G.,progression of heart lesions could help us to understand the

Zaher, S., Kassem, S. and Kotb, M. 1999. HLA class II associationsmechanism underlying autoimmunity in this pathology and with rheumatic heart disease are more evident and consistentalso result in a new way of therapy attempting to prevent the among clinically homogeneous patients. Circulation 99:2784.

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15 Smith, S. and Allen, P. 1992. Expression of myosin-class II majorhistocompatibility complexes in the normal myocardium occursAcknowledgementsbefore induction of autoimmune myocarditis. Proc. Natl Acad.Sci. USA 89:9131.We thank Drs P. Kourilsky, C. Pannetier and J. Even for providing us

with the Immunoscope software. The technical expertise of Mrs 16 Chopra, P., Narula, J., Kumar, S. A., Sachdeva, S. and Bathia,M. L. 1988. Immunohistochemical characterization of AschoffSandra M. Monteiro on flow cytometry experiments is acknowledged.

This work was supported by grants from the PADCT-CNPq no. 620087/ nodules and endomyocardial inflammatory infiltrates in left atrialappendages from patients with chronic rheumatic heart disease.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 Medicale Int. J. Cardiol. 20:99.17 Kemeny, E., Grieve, T., Marcus, R., Sareli, P. and Zabriskie, J. B.(FRM) and Association de la Recherche sur la Polyarthrite (ARP).

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