T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein

Original article

T cell epitope characterization in tandemly repetitiveTrypanosoma cruzi B13 protein

Lúcia C.J. Abel a, Leo K. Iwai a,d,e, Wladia Viviani h, Angelina M. Bilate a,d, Kellen C. Faé a,d,Renata C. Ferreira a,d, Anna C. Goldberg a,d,1, Luiz Juliano e, Maria A. Juliano e,

Bárbara Ianni b, Charles Mady b, Arthur Gruber g, Juergen Hammer f,2, Francesco Sinigaglia f,Jorge Kalil a,c,d, Edecio Cunha-Neto a,c,d,*

a Laboratory of Immunology, Heart Institute (InCor), University of São Paulo School of Medicine, Av. Dr. Enéas de Carvalho Aguiar, 44, Bloco II,9° andar, São Paulo, SP 05403-000, Brazil

b General Cardiopathies Division, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, SP 05403-000, Brazilc Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, SP 05403-000, Brazil

d Institute for Investigation in Immunology, Millennium Institutes, Brazile Department of Biophysics, Federal University of São Paulo/UNIFESP, São Paulo, Brazil

f Bioxell, Milan, Italyg Department of Pathology, Faculty of Veterinary Medicine and Zoothechny, University of São Paulo, São Paulo, Brazil

h Department of Biochemistry, Chemistry Institute, University of São Paulo, São Paulo, Brazil

Received 11 January 2005; accepted 29 March 2005

Available online 12 May 2005

Abstract

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan para-sites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas’ disease patients. Little isknown about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on Tcell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein byT cell lines, clones and PBMC from Chagas’ disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected indi-viduals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7),-DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope[FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Ystrain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides,molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolishedrecognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas’ disease patients, S15.4 waspreferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterizationof T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoanrepetitive antigens.© 2005 Elsevier SAS. All rights reserved.

Keywords: T cell epitope; B13 protein; Trypanosoma cruzi; Tandemly repetitive proteins; Immunodominant antigens

Abbreviations: ASY, asymptomatic T. cruzi infected individuals; CCC, chronic Chagas’ disease cardiomyopathy; CLIP class II-associated Ii peptide; CPM,counts per minute; EBV, Epstein–Barr virus; ECG, electrocardiogram; HA, hemmagglutinin; LPS, lipopolysaccharide; N or CTRL, non-infected controlindividuals; PBMC, peripheral blood mononuclear cells; PHA, phytohaemagglutinin; SI, stimulation index.

* Corresponding author. Tel.: +55 11 3069 5906/3069 5914; fax: +55 11 3069 5953.E-mail address: [email protected] (E. Cunha-Neto).

1 Department of Biochemistry, Chemistry Institute, University of São Paulo, São Paulo, Brazil2 Department of Genomic and Information Sciences, Hoffmann-La Roche Inc., Nutley, NJ, USA

Microbes and Infection 7 (2005) 1184–1195

www.elsevier.com/locate/micinf

1286-4579/$ - see front matter © 2005 Elsevier SAS. All rights reserved.doi:10.1016/j.micinf.2005.03.033

1. Introduction

Proteins containing regions of tandemly repetitive aminoacid sequences are present in several immunodominant pro-tein antigens from pathogenic protozoa, including the agentsof trypanosomiasis (Trypanosoma cruzi), malaria (Plasmo-dium spp.), toxoplasmosis (Toxoplasma gondii) and leishma-niasis (Leishmania spp.) [1–3]. The function of these regionsis still poorly understood. It has been suggested that they areinvolved in binding to repetitive structures within the para-site and to host cell receptors. Also, repetitiveness may havedefined physico-chemical or structural functions, as seen inthe case of collagen, myosin, heat shock proteins or glue pro-teins [4–6]. Despite the diversity in size, number and distri-bution of the repetitive elements, several distinctive featuresare held in common: the property of repetitiveness itself, thebias in the component amino acids (hydrophobic residuesrarely present), an unusual genetic and evolutionary history,and their immunodominance [1].

Little is known about the molecular mechanisms that leadto the immunodominance of repeated sequences, except fortheir multivalency which can make them activate B cellsdirectly and behave, in some cases, as T-independent anti-gens [1]. It is known that the repetitiveness of a variety ofagents causes T cell independent activation of B cells bycrosslinking hapten-specific surface immunoglobulin [7].There is much evidence that epitope conformation can be rel-evant for antigen–antibody binding [8,9], and antibodiesagainst native protein epitopes preferentially recognize pep-tide sequences with conformational preferences [10]. To-gether, these reports suggested that structural features—whichmay be present in repetitive epitopes—might play a criticalrole in protein immunodominance.

Several T. cruzi antigens contain tandemly repeated aminoacid motifs. The repetitive units already described in T. cruzihave a variety of different sizes and may vary from 5 to68 amino acids [11]. B13 is an immunodominant T. cruziantigen recognized by IgG serum antibodies from 98% ofT. cruzi infected individuals (both chronic Chagas’ diseasecardiomyopathy, CCC, and asymptomatic/indeterminate(ASY)) in Latin America and encodes a partially degeneratetandemly repetitive 12-amino acid motif present in a 140 kDamembrane protein [12]. In has been shown that the multiva-lent protein epitopes of T. cruzi B13 antigen [13] and Plas-modium falciparum CS protein [14], display a similar con-formation in each repetitive unit, leading authors tohypothesize that, at least in those cases, immunodominancewas linked to their ability to be recognized by a single type ofconformation-dependent antibody. Authors have speculatedthat amino acid sequences with conformational preferencesand increased antigenic potential may have been selected forin other immunodominant tandemly repeated antigenicdomains of protozoan proteins.

Even though evidence from some systems supports thatantibodies towards repetitive protozoan antigens may be Tcell-independent [1], it cannot be excluded that, even in these

cases, T cell help could boost antibody titers and affinity.Repetitive sequences in protozoan parasite antigens have alsobeen shown to elicit T cell dependent immune responses. Ithas been reported that peripheral T cells from P. falciparummalaria patients recognize T cell epitopes in EB200, a repeti-tive region of the P. falciparum antigen Pf332 [15,16], thePf155 repetitive domain of ring-infected erythrocyte surfaceantigen (RESA) [17] and the repetitive domain of circum-sporozoite CS protein [18]. However, so far, there is no infor-mation on fine characterization of T cell epitopes in proto-zoan tandemly repetitive antigens. In order to study whetherT cells from Chagas’ disease patients recognized immun-odominant B13 protein, we analyzed the PBMC response toB13 from Chagas’ disease patients. To finely characterize theT cell recognition of the tandemly repetitive units of immu-nodominant B13 protein, we analyzed the HLA class II bind-ing properties of B13 peptides and HLA association withPBMC responsiveness to B13, the minimal T cell epipopeand the HLA contact residues, and explored the putative TCRcontact residues. In order to search for differential recogni-tion of variant B13 epitopes, we analyzed the proliferativeresponses of PBMC from CCC, asymptomatic T. cruziinfected (ASY) and non-infected control individuals (N), aswell as a B13 epitope-specific T cell clone, against syntheticpeptides encoding naturally occurring Y strain T. cruziB13 variant sequence peptides. Our results identified fre-quent MHC-restricted T cell recognition of T cell epitopesfrom the repetitive domains in B13 protein.

2. Methods

2.1. Patients, materials and methods

Heparinized venous blood was obtained from Chagas’ dis-ease patients followed at the Heart Institute (InCor), Univer-sity of São Paulo School of Medicine. Chagas’ cardiomyopa-thy patients (CCC) fulfilled the following diagnostic criteria:positive serology for T. cruzi, typical ECG abnormalities (leftanterior hemiblock and/or right bundle branch hemiblock),varying degrees of ventricular dysfunction, with all othercauses of ventricular dysfunction/heart failure excluded.Blood samples were also collected from asymptomatic “inde-terminate” individuals (ASY), seropositive to T. cruzi, withnormal ECG and bidimensional echocardiography. Periph-eral blood samples were also collected from T. cruzi sero-negative, age and sex-matched normal volunteers (N) as acontrol group. Sample collection procedures have beencleared by the Internal Review Board of the University ofSão Paulo School of Medicine.

2.2. B13 protein

Recombinant T. cruzi B13 protein was obtained afterexpression of a recombinant clone obtained from a T. cruziYstrain expression library screened with sera from chronic Cha-

1185L.C.J. Abel et al. / Microbes and Infection 7 (2005) 1184–1195

gas’ disease patients [12]. The full amino acid sequence ofthe B13 insert in recombinant pMSgt11 plasmid is shown inTable 1 (GenBank accession number AY325808). The ex-pressed b−galactosidase–B13 fusion protein was purified onp-aminophenyl-b-galactopyranoside agarose columns.B13 recombinant protein contains 19 tandemly repeated12-amino acid motifs. Non-recombinant b-galactosidase (thefusion protein support) expressed from intact pMSgt11 plas-mid was processed similarly and used in some experimentsas a negative control. Endotoxin-depleted B13 proteinobtained by treatment with Polimyxin B-Sepharose 4B (SigmaChemical Co., St. Louis, MO, USA) was added to selectedwells in control experiments [19].

2.3. Synthetic peptides

13-mer overlapping synthetic peptides encompassing thewhole 12-residue B13 motif including some sequence vari-ants [12,20,21], as well as 10, 15-mer peptides containingthe variant B13 sequences and centered HLA-DQ7-bindingframes, from B13 protein (Genbank accession numberAY325808.1) were obtained with Fmoc solid-phase chemis-try [22] using multiple peptide synthesizers from AdvancedChemTech (model 396, Louisville, KY, USA) and Shimadzu(model PSSM-8, Tokyo, Japan). Overlapping 15-mer B13peptide mixtures containing degenerate variant positions, wereobtained with combinatorial chemistry by the addition of twoor three fmoc-derivatized amino acid residues at appropriatesteps during automated peptide synthesis. The indicator pep-tides GFKA7, GYRA2YA4, IAYDA5, UD4, HA, CLIP 89-101(class II-associated Ii peptide) and TT 830-843 [23] wereN-terminally biotinylated before cleaving them from resin bysequentially coupling two 6-aminocaproic acid spacers on the

N-terminus and one biotin molecule, using the above-described standard procedure. Peptides were analyzed byMALDI-TOF mass spectrometry (Tof-Spec E, Micromass,Manchester, UK) and by analytical reverse-phase HPLC (Shi-madzu, Tokyo, Japan) and were routinely > 80% pure.

2.4. Binding of peptides to HLA class II molecules

MHC class II molecules were used from the followinghuman HLA-homozygous lymphoblastoid cells: DR1(DRB*0101) from HOM-2; DR2 (DRB1* 1501 +DRB5*0101), DR3 (DRB1*1701) from WT49; DR4(DRB1*0401) from BSM; DR5 (DRB1*1101) from SWEIG;DR7 (DRB1*0701) from EKR and DR8 (DRB1*0801) fromBM9. DR2 from transfected cells (DRB1*1501) was iso-lated from the transfectant cell line L466.1, a kind gift formDr. R.W. Karr (Monsanto, St Louis, MO, USA). HLA-DRand -DQ Molecules were affinity-purified as described[24,25]. HLA-DQ7 (DQA1*0501/DQB1*0301) was puri-fied from SWEIG lymphoblastoid cells using monoclonalantibody SPV-L3 [25]. B13 peptide-binding assays were per-formed with HLA molecules in high throughput competitivelabeled-peptide ELISA-based assays [23]. Briefly, seriallydiluted peptides were incubated in the presence of N-terminalbiotinylated indicator peptides as described and affinity-purified MHC class II molecules (100 ng protein) in a pro-tease inhibitor cocktail, and transferred to transferred to anELISA plate coated with monoclonal antibodies anti-HLA-DQ SPV-L3 or anti-HLA-DR L243 (ATCC HB55),incubated with streptavidin-alkaline phosphatase, and4-nitrophenylphosphate. Data are scored as the concentra-tion of test peptide capable of inhibiting 50% of the bindingof labeled-peptide to that HLA class II molecule (IC50%).

Table 1Binding of synthetic B13 peptides scanning two 12-mer B13 tandemly repetitive units to different HLA class II molecules

1186 L.C.J. Abel et al. / Microbes and Infection 7 (2005) 1184–1195

Identification of B13 peptide contact residues to DQ7(DQA1*0501/DQB1*0301) was performed using singlelysine substitutions or double valine and histidine substitu-tions because they lack non-specific inhibitory effects on bind-ing to HLA-DQ7 [23].

2.5. HLA class II typing

DNA was extracted alternatively by DTAB/CTAB or salt-ing out methods [26]. DR typing was performed by low reso-lution PCR-SSP [27] as previously described [28]. DQA1 andDQB1 typing were performed by PCR-SSO using genericprimers for exon-2 amplification [26].

2.6. Peripheral blood mononuclear cell (PBMC)proliferation assays

Peripheral blood mononuclear cells (PBMC) were iso-lated from peripheral blood by Ficoll–Hypaque density gra-dient centrifugation (d = 1.077), washed and incubated in Dul-becco’s modified Eagle’s medium (DMEM-GIBCO, GrandIsland, NY, USA) supplemented with 2 mM L-glutamine,1 mM sodium pyruvate, 50 µg/ml gentamicin, 10 mM HEPESbuffer and 10% normal human serum (complete medium). Inthe proliferation assay, cell cultures from CCC, ASY and Nindividuals were carried out in triplicate in 96-well-flat-bottom culture plates (105 cells per well; final volume 0.2 ml)with B13 protein (5 µg/ml) or B13 15-mer peptides (25 µM);phytohemagglutinin (PHA, 5 µg/ml; Sigma) was used as posi-tive control, and complete medium as a negative control.b-galactosidase or endotoxin-depleted B13 protein was addedto selected wells. Ideal concentrations for PBMC prolifera-tion assays were previously established from dose-responsecurves for recombinant B13 protein [29] and 15-mer B13 pep-tides [30]. Plates were incubated in 5% CO2 at 37 °C for 5 daysand cultures were pulsed with 1 µCi per well [3H]-thymidine(Amersham, Buckinghamshire, UK) for the final 18 h.[3H]-thymidine incorporation was measured at the Betaplatebeta counter (Wallac Inc, Turku, Finland). Data are repre-sented as the stimulation index (SI) defined as mean CPMexperimental triplicates with antigen/mean CPM of tripli-cates of culture medium control. SI values ≥ 2.0 were consid-ered positive.

2.7. Antigen-specific T cell lines and clones

B13- and B13 peptide-specific T cell lines and clones wereestablished from the peripheral blood of HLA-DQ7+ indi-viduals (with a previous positive PBMC proliferative responseto B13), by the addition of B13 protein (5 µg/ml) or B13 pep-tides (25 µg/ml) and incubation in complete medium contain-ing IL-2 essentially as described [25]. The lines wereexpanded by re-stimulation every 15 days with phytohemag-glutinin (PHA, 5 µg/ml), 40 U/ml IL-2 and irradiated(5000 rads) PBMC (106 well) and subjected to proliferationassays 10 days after the last PHA stimulus. Peptide-driven T

cell lines were also cloned by limiting dilution essentially asdescribed [31], with the addition of IL-7 and IL-15 (5 ng/ml)in the expansion phase. Proliferation assays of peptide-driven T cell line and clone were carried out incubating 3 ×104 T cell line and 105 irradiated HLA-DQ7 allogenicAPC/well in triplicate in a 96-well-U-bottom culture plates(final volume 0.2 ml) with B13 protein (5 µg/ml) or B1315-mer peptides (25 µM) and PHA at 5 µg/ml as a positivecontrol (Sigma Chemical Co., St Louis, MO, USA). Data arerepresented as stimulation index (SI) defined above. SI val-ues ≥ 2.0 were considered positive.

2.8. Mapping of the minimal B13 T cell epitope withcombinatorial peptide libraries

As an approach to mapping the T cell epitope in the degen-erate 12-mer B13 repeats, we synthesized twelve 15-mer pep-tides overlapping with a 1-residue step and tested their rec-ognition by a B13-specific T cell line. To allow forrepresentation of all sequence variants reported in B13 andB13-like sequences [12,20,21], each 15-mer peptide was madedegenerate in the variant positions, which was obtained, withcombinatorial chemistry, by the addition of two or three fmoc-derivatized amino acid residues at appropriate steps duringautomated peptide synthesis [32]. Each 15-mer B13 mixture-based peptide library had 7–10 degenerate positions, with288–5184 different sequences. Such “complex” mixture pep-tide libraries were diluted in culture medium, filtered and usedat 250 µM total concentration; the concentration for each indi-vidual sequence was 0.05–1.00 µM in different mixtures.Fifteen-mer overlapping restricted combinatorial peptidelibraries including only the variant sequences which werecommonly encountered in all B13 and B13-like sequences[12,20,21] were also synthesized, and each “common” 15-merB13 restricted combinatorial peptide library had 8–32 differ-ent sequences. “Common” restricted combinatorial peptidelibraries were used at 20 µM total concentration, and the con-centration for each individual sequence was 0.6–2.4 µM indifferent mixtures. Peptides were preincubated for 2 h withthe irradiated (10,000 rad) EBV-B cell line SWEIG, which ishomozygous to HLA-DQA1*0501/DQB1*0301. The B13-driven T cell line from HLA-DQ7+ CCC patient SMC wasincubated for 96 h in 5% CO2 at 37 °C, in triplicates of aflat-bottom 96 well culture plate, irradiated SWEIG EBV cellspulsed with the two series of 15-mer overlapping peptidelibraries, and cultures were pulsed with 1 µCi per well[3H]-thymidine (Amersham, Buckinghamshire, UK) for thefinal 18 h. [3H]-thymidine incorporation was measured withthe Betaplate beta counter (Wallac Inc, Turku, Finland).

2.9. Molecular modeling of the HLA-B13 peptidecomplexes

Model building, visualization and quantitative study byMolecular Mechanics and Dynamics were carried out in anIBM workstation RS6000- 43P, using the INSIGHT-II pack-


age (MS Inc., San Diego, USA). All potential energy evalu-ations were performed using CVFF forcefield, considering apH=7.2 and a dielectric constant e = 80 in order to simulateimplicitly the aqueous surroundings. Potential energy mini-mizations were carried until the energy derivative value wasup to dE = 0.05 kcal/mol*A. The primary sequence of HLA-DQA1*0501/DQB1*0301 (DQ7) was manually aligned tothe HLA-DR1 sequence and used for homology building(1DLH [33]). The resulting model was energy minimized anddirectly used for complexation of the 13-mer B13 peptideused for HLA-DQ7 binding assay: SPFGQAAAGDKPS. Thedocking step consisted in transposing the HLA-DR1-complexed HA peptide main chain coordinates to theB13 peptide, since both are expected to have a polyproline-like helix conformation. The template mouse hemoglobinHb(64–76):Lys9 peptidic residue [34] was used as an uniquereference point for B13 alignment, since the latter Lys11 resi-due can be expected to form a salt bridge with the HLA con-served aspartic residue: HLA-DQ7b:Asp55, correspondingto the pair Hb:Lys9/DR1b:Asp57. Therefore, the B13 modelwas fitted into the HLA-DQ7 model groove with an orienta-tion analogue to the one exhibited by the template complex.The resulting HLA-DQ7-B13 peptide complex was thenrefined by a T = 400 K, t = 350 ps. Molecular dynamics simu-lation (including 50 ps for thermal equilibration), so that theoverall structure and namely peptidic side chains and proteinreceptacle were allowed to mutually adapt. The mean struc-ture assumed by the complex during this step was the onesubmitted to a simulated annealing experiment: starting at300 K, lowering to 100 K, and then to 50 K, staying for atotal of 65 ps (including 15 ps for thermal equilibration) ateach temperature. The final structure was then energy opti-mized, and constitutes the refined HLA-DQ7/B13 peptidemodel. Models for each of the 10, 15-mer B13 peptide vari-ants could be built with basis on the above-described HLA-DQ7/13-mer antigen model, by adding two residues to theoriginal peptide and substituting, where needed, the originalresidues according to each variant sequence. New structureswere then allowed to adjust by geometry optimization, con-sidering at first a semi-rigid supermolecule where only thepeptide substituted side chains were unfrozen, and thereforeable to relax; then relaxing all peptide atoms, still keeping allprotein atoms frozen; finally, submitting the fully relaxed com-plex structure to a molecular dynamics plus SimulatedAnneal-ing refinement. All these steps have been carried followingthe same protocol as reported above for the 13-mer peptidecomplex.

2.10. Data analysis

Mann–Whitney’s rank sum test was used to compare con-tinuous variables. Fisher’s exact test was used to comparefrequencies of responder and non-responder individuals in Tcell proliferation assays.

3. Results

3.1. T cell response against B13 protein: HLA restriction

We found that PBMC proliferation to recombinant proteinB13 was similar in frequency and intensity between the CCCandASY groups (56% and 43% responders, respectively) and,surprisingly, even among the normal control individuals(60%). Average stimulation indexes for CCC, ASY and Ngroups were 4.8 ± 6.2, 3.1 ± 5.1 and 3.8 ± 4.4, respectively.PBMC from N individuals that responded to B13 proteinfailed to respond to b-galactosidase, the fusion protein sup-port, and LPS depletion of recombinant B13 with immobi-lized polimyxin B failed to decrease B13-induced prolifera-tion among N PBMC, ruling out the recognition of the supportprotein or contaminating bacterial products (data not shown)[28]. Table 1 shows that overlapping 13-mer B13-derived pep-tides scanning one 12-mer repetitive unit can bind, albeit withlow affinity, to HLA-DQ7 (DQA1*0501/DQB1*0301) andHLA-DR1 (DRB1*0101) molecules. Peptides that shared theKP(S/P)(L/P)FGQAAG sequence also showed low affinitybut significant binding to EBV-derived DR2b (DRB1*1501/DRB5*0101, but not to DR2a (DRB1*1501) derived fromtransfectant cell L466.1, indicating that DR51 (DRB5*0101)may be able to bind B13 peptides. The specificity of bindingto HLA-DQA1*0501/DQB1*0301 could be observed by thelack of detectable binding to DR5 (DRB1*1101) present inthe same lymphoblastoid cell line from which the DQ7 mol-ecule was extracted (SWEIG). Several different 13-mer pep-tides starting at different positions of the B13 tandem repeatcan bind to HLA molecules, with the single exception of thepeptide QAAAGDKPSLFGQ. It should also be mentionedthat peptides in the third to fifth row, representing the com-mon sequence variants of B13 [12,20,21], along with othertested variants (data not shown) bind with similar affinity tothe HLA molecules. The full sequence of the B13 proteininsert with its 19 partially degenerate tandem repeat units isalso shown. Analyzing the HLA profile of individuals show-ing PBMC proliferative responses to B13 protein (Fig. 1), weobserved that 84% of B13 responders carry at least one of theDR1, DR2, or DQA1*0501 B13-binding HLA alleles shownin Table 1, a statistically significant difference when com-pared to 65% among B13 non-responders (P = 0.048). Takenindividually, DQA1*0501 was also significantly more repre-sented among B13 responders than non-responders(DQA1*0501: 53% vs. 28%, P = 0.03); among Chagas’ dis-ease patients, the difference in frequencies of DQA1*0501among responders and non-responders was even more promi-nent (55% vs. 15%, P = 0.001), confirming its major role inT cell presentation of B13 epitopes (Fig. 1).

3.2. Mapping of the minimal DQ7-restricted B13 T cellepitope

HLA DQA1*0501 is the most frequent B13-binding HLAallele in the general population—half of the times paired to


DQB1*0301, as stated above. Given the availability of theHLA-DQA1*0501/DQB1*0301 (HLA-DQ7) molecule forpeptide-binding assays, we studied the minimal epitope rec-ognized in the context of HLA-DQ7. Table 2 shows the pro-liferative response of B13-specific T cell line from HLA-DQ7+ CCC patient SMC to the two series of 15-meroverlapping library peptides degenerate at variant positions,one representing all possible variant positions (peptide librar-

ies 1–12) and the second one only representing variantscommon to all isolates (peptide libraries 13–24), presentedby homozygous EBV-B line SWEIG (DRB1*1101,DQA1*0501/DQB1*0301). The anti-B13 T cell line SMCrecognized all 15-mer “complex” peptide libraries sharing the10-mer sequence FGQAA(A/E)(G/A)(D/E)(K/R)(P/L), withan average stimulation index of 6.6 ± 0.3; negative peptidesshowed an average stimulation index of 1.1 ± 0.3 (Table 2).All 15-mer “common” peptide libraries including the 10-merFGQAAAG(D/E)KP sequence, contained in the recognized“complex” peptide libraries, were recognized with an aver-age stimulation index of 8.0 ± 0.1; negative peptides showedan average stimulation index of 1.4 ± 0.3 (Table 2).All 15-merpeptide libraries bound detectably to HLA-DQ7 (2–40 µMIC50% range, data not shown). B13 peptide-pulsed EBV-Blines WT-49 (DRB1*0301, DQA1*0501/DQB1*0201) andPRIESS (DRB1*0401, DQA1*0301/DQB1*0302) failed tostimulate anti-B13 T cell line SMC (data not shown), thusconfirming the HLA restriction of the response. Results indi-cated that the HLA-DQ7 restricted minimal T cell epitope inB13 (FGQAAAG(D/E)KP) was a common sequence, presentin 11 out of the 19 sequenced repetitive elements present inYstrain B13 protein as in Table 1. In the following experi-ments, we numbered B13 peptide sequences according to theminimal epitope, with the first residue (F) appearing as resi-due F1, and the C-terminal (P) as P10. For longer peptidesresidues extending from the N-terminal end had negative num-bers.

3.3. Identification of critical HLA-DQ7 contact residues inB13 peptides

Lysine-substituted analogues of B13 peptide SPF-GQAAAGDKPS, comprised between residues S(-2) and S11,and containing the minimal DQ7-restricted B13 T cell epitope,were subjected to binding assays to HLA-DQ7. Table 3A indi-cates that lysine substitution of residues F1, G2 and A4 abol-ish peptide-binding to HLA-DQ7(SPFGQAAAGDKPS),while substituting residue A5 partially inhibits peptide-binding. In order to confirm these findings, we tested the bind-ing of a B13 peptide in a different frame, comprised betweenD(-5) and D8 (DKPSPFGQAAAGD) and its valine-histidinedouble-substituted (VxH) analogues to HLA-DQ7. Table 3Bshows that the double substitution of residues G2 and A4(DKPSPFGQAAAGD) completely abolished binding. Fur-ther in support of this finding, Table 1 shows that the onlypeptide which failed to bind to HLA-DQ7 was the peptidecomprised between Q(-10) and Q3, QAAAGDKPSLFGQ,devoid of the G2-Q3-A4 sequence. Together, data support thenotion that G2 and A4 are the small-side chain major contactresidues to HLA-DQ7, positioned at relative positions i and i+ 2, in agreement with previously published data showingthat DQ7-binding residues possess small-side chains and arelocated in the central part of an epitope [23], with a minorcontribution for HLA-binding from residues F1 and A5.

Fig. 1. Proliferative response (105 cells per well) to B13 protein (5 µg/ml) inCCC (n = 27), ASY (n = 41) and normal control individuals (n = 23) sortedaccording to the expression of B13-binding HLA class II allelesDQA1*0501(DQB1*0301 or DQB1*0201), DR1/DR2, or none of them.Each point represents the SI value of a single individual. A few individualscarried both DQA1*0501 and DR1 or DR2 and were therefore depicted aspoints in both sections of the graph.

Table 2Proliferative response of anti-B13 T cell line SMC to overlapping 15-mermixture peptides presented by HLA-DQ7+ EBV line SWEIG


3.4. PBMC recognition of 15-mer natural variant Y strainT. cruzi B13 peptides among clinical groups

Native 13-mer B13 peptides used in binding assays(Table 3A) or the 12-mer PFGQAAAGDKPS containing theminimal epitope were poor stimulants in PBMC prolifera-tion assays, inducing responses in less than 15% of HLA-DQ7+ individuals that responded to recombinant B13 pro-tein (data not shown). The mentioned peptides had the G2-Q3-A4 binding cassette off-center, and lacked N-terminal flankingresidues that could be important for T cell recognition [35].Furthermore, as the 12-mer tandemly repetitive unit ofB13 protein occurs in 19 imperfect copies, we synthesized10 singular 15-mer B13 peptides (Fig. 2C), including aG2-Q3-A4 DQ7-binding motif positioned in the central regionof the peptide and encompassing all natural sequence vari-ants found in recombinant B13 protein from T. cruzi Y strain(see Table 1). We tested the recognition of the 10 singularB13-derived peptides by PBMC from HLA-DQ7 positiveB13-responsive, HLA-DQ7-positive CCC and ASY patients,and normal controls (Table 4). Of the 31 B13-responsive indi-viduals tested, 90% responded to at least one B13 peptide.The proliferative response to B13 peptides in most individu-als in the CCC and N groups was similar in magnitude to thatinduced by recombinant B13 protein, while among ASYpatients the response to B13 peptides used to be much lowerthan that directed to B13 protein. Similarly, CCC patients andN individuals recognized a significantly higher number of pep-tides than ASY patients (average of 3.2, 3.1 and1.5 peptides/individual, respectively; CCC vs. ASY or N vs.ASY, P < 0.05). Profiles of peptide recognition varied signifi-cantly: out of 28 individuals recognizing any peptide, we

found 26 distinct combinations of the 10 B13-derived pep-tides recognized by single individuals (Table 4). Analyzingpeptide reactivity according to clinical groups, we observedthat peptide S15.9 was the most frequently recognized byCCC and ASY patients (71% and 63%, respectively), andhad the highest average stimulation indexes among Chagas’disease patients (4.6 and 3.5 by CCC and ASY, respectively)but was not recognized by normal individuals. PeptideS15.7 was the most frequently recognized among B13-responsive, HLA-DQ7-positive normal individuals (55%).Peptide S15.4 (KPPPFGQAAAGDKPP) was frequently rec-ognized by CCC patients, and threefold less recognized byASY (64% vs. 18%, P = 0.07). Peptides S15.1 and S15.3 werethe only ones more frequently recognized by ASY (27% and46%) than CCC patients (9% and 18%) (P = 0.6 and P = 0.3,respectively) (Table 4 and Fig. 3).

3.5. Recognition of B13 variant peptides by S15.4-specificT cell clone

Proliferation assays with S15.4-specific T cell clone3E5 against the other 15-mer B13 protein variant peptidesshowed that, at least for this T cell clone, positions p-3 to p-1,p7 and p11 maybe important HLA/TCR contact residues asamino acid substitutions in these positions abolished the rec-ognition (Table 5).

3.6. Molecular modeling of the B13 peptide:HLA-DQ7 complex

Molecular modeling of complexes between HLA-DQ7 andB13 peptides were performed in order to compare structuraland functional (i.e. peptide-binding assays and T cell recog-nition) data. Molecular modeling of the 13-mer B13 peptideSPFGQAAAGDKPS, subject to the lysine substitution-DQ7 binding assay analysis, complexed to HLA-DQ7 showedthat the region F1 to K9 fits into the peptide-binding groove.Side chains of residues at relative positions F1, G2, A4, A5,D8 fit into pockets in the antigen-binding groove, either atthe a-helical portion (F1) or to its floor; G2 fits a shallowerpocket allowing direct contact of the peptide main chain tothe groove. It is of note that molecular modeling confirmedall four major and minor positions of HLA contact residues(F1, G2, A4, A5) identified by the lysine-substituted B13 pep-tide-binding assay. Interestingly, both major and minor HLAcontact residues are conserved among all 19 sequenced repeti-tive elements present in Y strain B13 protein (Table 1).Molecular modeling of a complex between the B13 variantpeptide S15.4 comprised between residues K(-4) and P11(KPPPFGQAAAGDKPP) and HLA-DQ7 showed again thatthe region F1 to K9 fits into the peptide-binding groove (topview of peptide:HLA-DQ7 model, Fig. 2A). Solvent-exposedresidues rendered in green were F1, G2, A6, K9, P11. Theflanking N-terminal region [P(-2) to K(-4)] lies far from thepeptide-binding groove, and the resulting high mobility pre-cludes the attribution of a single energetically favorable side

Table 3HLA-DQ7 binding assay of substituted analogues of B13 peptides for theidentification of HLA-DQ7 binding residues in B13. A. Single (lysine)-substituted analogues and B. Double-(valine_histidine) substituted analo-gues


chain configuration, but it can potentially interact with thesolvent and the T cell receptor-particularly position -2(Fig. 2A). Among the 10 natural sequence variants 15-merB13 peptides docked to DQ7 tested above, several solvent-exposed (and therefore TCR-exposed) positions also showsequence variation, like positions 6 (A/E) and 11 (P/S/A),along with the apparently TCR-exposed position -2 (P/S/A)and potentially TCR-exposed position -3 (P/L) at theN-terminus region (Fig. 2A, B). Among the 10 variantB13 peptides, seven distinct six-residue solvent-exposedsequences are displayed, corresponding to each combinationof polymorphic sequences at the exposed positions (Fig. 2C),with corresponding changes in the TCR-exposed side chains(Fig. 2A–C).

4. Discussion

In this paper, we have finely characterized the T cellepitopes in protozoan tandemly repetitive antigen B13. Wehave shown that the tandemly repetitive sequences of T. cruziB13 protein can be presented for T cell recognition in the

context of at least three distinct HLA class II molecules(Table 1 and Fig. 1), and identified the minimal T cell epitoperecognized in the context of the most frequent of them, HLA-DQ7 (Table 2). Furthermore, we identified its HLA-DQ7 con-tact and putative TCR-exposed residues (Table 2 and Fig. 2),and demonstrated that variant B13 T. cruzi epitopes are dif-ferentially recognized between CCC and ASY patients(Table 4 and Fig. 3).

The finding that common sequence variants of B13 pro-tein peptides bind with similar IC50% values to HLA-DRB1*0101, DRB5*0101 DQA1*0501/DQB1*0301 indi-cates that each B13 protein unit can generate several HLA-binding epitopes. The low IC50% values observed in HLA-binding assays with peptides from tandemly repetitiveB13 protein (Table 1) are in line with similar findings for theT1 peptide from the tandem repeats of P. falciparum CS pro-tein [36] and may be related to their biased amino acid usage,favoring small and charged side chain residues [1]. The find-ing that such HLA alleles are significantly more representedamong patients who show PBMC proliferative responses torecombinant protein B13 (Fig. 1) demonstrates that the B13-

Fig. 2. Molecular modeling of B13 peptides docked to the antigen-binding groove of the HLA-DQ7 molecule after homology building with the HLA-DR1:HApeptide complex (1DLH [33]) as described in Section 2. A. Top view of complex between HLA-DQ7 and 15-mer B13 peptide S15.4. B13 peptide (white) andDQ7 molecule (blue) rendered as Van der Waals contours, peptide main chain without hydrogens displayed as red sticks, F1, Q3, A6, K9, P11 and A8 TCR-exposed side chains with hydrogens, in green; putative N-terminal proline residues rendered in red with hydrogens. B. Side view of all 10 superimposedB13 variant peptides rendered as sticks on the antigen-binding groove of HLA-DQ7 molecule. Each peptide is rendered in a different color. Polymorphicpositions are identified in each peptide. C. Alignment of B13 variant peptides, their TCR contact positions, and TCR contact sequence patterns.


binding HLA class II molecules can indeed presentB13 epitopes to T cells. A parallel study showed that PBMCfrom 92% of the individuals carrying the B13-binding HLAalleles presented proliferation, IFN-c or IL-4 productionresponses to recombinant B13 protein (data not shown).Together with the fact that 80% of individuals in the generalpopulation carry at least one of the B13-binding HLA alleles[28], results suggest that essentially all individuals carryingone of the B13-binding HLA alleles, or the majority of thepopulation, can be taken as B13 responders.

The identification of the 10-mer sequence FGQAAAG-(D/E)KP as the minimal DQ7-restricted T cell epitope(Table 2), which contains the major antibody epitopeFGQAAAGDK [37,38], may have implications in cognate T:B cell interactions. The identification of glycine and alanineas being the major contact residues of the B13 repetitivesequence peptides with HLA-DQ7, as reported by the substi-tution assays (Table 3A, B) is in line with published data forboth DQ7 (DQA1*0501/DQB1*0301) [23] or DQA1*0301/DQB1*0301 [39]. The fact that the four major and minor HLA

contact residues are conserved among the sequenced repeti-tive elements present in Y strain B13 protein (Table 1), indi-cates that B13 sequence polymorphism does not impair theability of sequence variants to bind to HLA-DQ7. The factthat PBMC samples from 90% of B13-responsive tested indi-viduals showed proliferative responses to at least one of the15-mer variant B13 peptides, with magnitudes similar to thoseagainst recombinant B13 protein (Table 4) suggests that wehave identified the major DQ7-restricted T cell epitopes ofB13 protein. Several consistent differences were observed atthe level of recognition of individual B13 peptides. The factthat two variant B13 peptide epitopes S15.9 and S15.10 (Fig. 3and Table 4) were exclusively recognized by Chagas’ diseasepatients, indicate that in vivo presentation of B13 protein alonginfection was necessary for the appearance of the reactivi-ties. Peptide S15.9 is probably the major “public” T cellepitope of B13 protein, since it is the most frequently recog-nized and with the highest stimulation indexes between bothCCC and ASY patients. The fact that peptide S15.4 was rec-ognized more frequently by CCC than ASY patients suggests

Table 4Proliferative response of HLA-DQ7+ CCC, ASY and N individuals to recombinant B13 protein and variant B13 peptides


that differential recognition of the peptides may be clinicallyrelevant, given the demonstrated T cell cross-reactivitybetween B13 protein and cardiac myosin [40–42]. Recent datafrom our group has shown that a S15.4 specific T cell clonecan cross-reactively recognize cardiac myosin epitopes [42].One cannot exclude, however, that the differential recogni-tion of B13 peptides is related to sequence polymorphisms ofthe T. cruzi B13 gene itself. The fact that the S15.4 can onlybe found in B13 from the Y strain, but not among availableB13-like sequences from other T. cruzi strains [20,21] maysuggest that selective recognition of S15.4 by CCC patientscould be associated with infection by distinct T. cruzi strainsbearing the respective epitopes. Similarly, one could notexclude that the low recognition by ASY patients of theB13 peptides tested here could be secondary to infection by astrain with different sequences. On the other hand, the simi-lar PBMC proliferative responses to both recombinant

B13 protein and B13 variant peptides S15.1 to S15.8 amongChagas’ disease patients and normal individuals (Table 4 andFig. 3) indicates that individuals not exposed to T. cruzi aresensitized to sequences similar to B13 protein. This is in linewith previous reports that non-exposed individuals mayrespond to antigens from different pathogens such as P. fal-ciparum [43], Leishmania [44], along with another T. cruziantigen [45] in primary T cell proliferation assays. In the caseof B13, the possibility of its being a superantigenic effect isdismissed by the recognition of B13-derived synthetic pep-tides by N PBMC (Table 4 and Fig. 3).

Molecular modeling and molecular dynamics of the HLA-DQ7:S15.4 B13 epitope complexes (Fig. 2A, B) indicatedthat certain solvent-exposed positions (p-1, p1, p3, p6, p9 andp11) are variant. Given the fact that amino acid variation inpositions p-3 to p-1, p6, p7 and p11 (Table 5) abrogated rec-ognition of S15.4-specific T cell clone 3E5, it is likely thatamino acids at some of these positions are important T cellcontact residues. Results from the B13 S15.4 peptide-specific T cell clone with lysine-substituted S15.4 peptides[42] are consistent with the observed results, indicating thatresidues at N-terminal region (p-3 to p-1) and at positionsp6 and p11 are important T cell contact residues. The factthat 12-mer or 13-mer peptides (Table 3A) containing theminimal B13 epitope (Table 2) but lacking N-terminal flank-ing residues were not as efficiently recognized as the 15-merB13 peptides (Table 4) by PBMC from HLA-DQ7+ individu-als (data not shown) suggests that N-terminal flanking resi-dues may also include important solvent-exposed TCR con-tacts. Assuming position p-2—not hindered by the HLA-DQ7 molecule—is another TCR contact, we have threepolymorphic positions (p-1, p6 and p11) out of the six TCR-exposed sites (p-1, p1, p3, p6, p9 and p11).

In summary, we demonstrated for the first time down tostructural detail that each tandemly repetitive T. cruzi B13 pro-tein possesses several variant T cell epitopes that can be rec-ognized in an MHC-restricted manner. Strikingly, MHC-contact residues are conserved in all repetitive regions, whilesolvent-exposed ones include variant positions, an ideal situ-ation for T cell recognition by genetically heterogeneoushosts.Additionally, the increased molar concentration of indi-vidual B13 epitopes may compensate the low-avidity bind-ing to HLA molecule. Thus, in spite of the detected low-avidity binding of B13 peptides to HLA class II molecule,we observed frequent T cell responses to B13 epitopes. How-ever, we do not know yet whether this is a general mecha-nism that may be associated with antibody immunodomi-nance of protozoan proteins or whether it was peculiar to theB13 protein. B13-derived 15-mer epitopes were shown to beantigenic and elicited peptide-specific T cells with prolifera-tive capacity, usually associated with central T cell memory.These peptide-specific cells can hypothetically provide T cellhelp to B cells for antibody production, even in the case ofrepetitive antigens. To test the hypothesis that this is a gen-eral phenomenon, defined T cell epitopes in the repetitiveregions of other immunodominant tandemly repetitive proto-

Fig. 3. Proportion of responders to 15-mer variant B13 peptides among HLA-DQ7+ individuals responsive to recombinant B13 protein. A. CCC vs. ASYpatients. B. Chagas’ disease vs. normal controls. Percent responders for agiven peptide in a clinical group = number of individuals displayingSI ≥ 2.0/number of individuals in group.


zoan antigens should be searched for, perhaps with the use ofcombinatorial peptide libraries.

Acknowledgments

This work has been supported by grants from São PauloState Research Foundation (FAPESP) 94-1206-0 and96/1440-7, Brazilian National Research Council (CNPq)520533/97 (E.C.N.) and the Howard Hughes Medical Insti-tute (J.K.). We thank Sandra Drigo for help in the HLA typ-ing of critical samples.

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T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein