Locally Produced Survival Cytokines IL-15 and IL-7may be Associated to the Predominance of CD8+ Tcells at Heart Lesions of Human Chronic ChagasDisease Cardiomyopathy

S. G. Fonseca*,�, M. M. Reis�, V. Coelho*,�, L. G. Nogueira*,�, S. M. Monteiro*, E. C. Mairena*,F. Bacal§, E. Bocchi§, L. Guilherme*,�, X. X. Zheng–, F. Y. Liew**, M. L. Higuchi�, J. Kalil*,�,��& E. Cunha-Neto*,�,��

Introduction

Chronic Chagas disease Cardiomyopathy (CCC) is the mostimportant clinical consequence of the infection by theprotozoan intracellular parasite Trypanosoma cruzi, affecting16–18 million individuals in Latin America. T. cruzireplicates in the cytoplasm of host cells and establishes achronic, low-grade, lifelong infection. CCC is an inflamma-tory-dilated cardiomyopathy that may lead to a fatalcourse, affecting around 10–30% of infected individuals

5–30 years after the primary infection. The remaining60–70% chronically T. cruzi-infected individuals remainasymptomatic for many years. Histopathologically, CCC ischaracterized by a diffuse myocarditis with focal aspects; amononuclear infiltrate, intense heart fibre damage, promin-ent fibrosis and scarcity of T. cruzi parasites [1–3].Increased local expression of the cytokines IFN-c andTNF-a [4, 5], interleukin (IL)-6 and IL-4 [5], as well asHLA class I and II molecules and adhesion molecules hasbeen reported [6]. Real-time PCR analysis showed that the

*Laboratory of Immunology, Heart Institute of

the Sao Paulo University Medical School, Sao

Paulo, Brazil; �Institute for Investigation in

Immunology, Millennium Institutes, Sao Paulo,

Brazil; �Laboratory of Pathology, Heart

Institute of the Sao Paulo University Medical

School, Sao Paulo, Brazil; §Heart Failure and

Heart Transplantation Unit, Heart Institute of

the Sao Paulo University Medical School, Sao

Paulo, Brazil; –Division of Immunology, Beth

Israel Deaconess Medical Center, Harvard

Medical School, Boston, MA, USA; **Division

of Immunology, Infection and Inflammation,

Glasgow Biomedical Research Centre,

University of Glasgow, Glasgow, UK; and

��Division of Clinical Immunology and Allergy,

University of Sao Paulo, School of Medicine

Sao Paulo, Brazil

Received 13 April 2007; Accepted in revisedform 24 May 2007

Correspondence to: Dr E. Cunha-Neto,

Laboratorio de Imunologia, Instituto do

Coracao, Universidade de Sao Paulo. Av Dr

Eneas de Carvalho Aguiar, 44 BL 2, 9º andar,

Sao Paulo, SP 05403-000, Brazil. E-mail:

edecunha@gmail.com

Abstract

Human chronic Chagas disease cardiomyopathy (CCC) is an inflammatory-dilated cardiomyopathy occurring years after infection by the protozoanTrypanosoma cruzi. The heart inflammatory infiltrate in CCC shows a 2:1predominance of CD8+ in relation to CD4+ T cells, with a typicalTh1-type cytokine profile. However, in vitro expansion of infiltrating T cellsfrom heart biopsy-derived fragments with interleukin-2 (IL-2) and phyto-haemagglutinin leads to the outgrowth of CD4+ over CD8+ T cells. Wehypothesized that survival cytokines, such as IL-2, IL-7 and IL-15 mightbe differentially involved in the growth and maintenance of heart-infiltra-ting and peripheral CD8+ T cells from CCC patients. We found that IL-7and IL-15 were superior to IL-2 in the expansion and viability of CD8+

T cells from both PBMC and heart-infiltrating T-cell lines from CCCpatients, and the combination of the three cytokines showed synergiceffects. Heart-infiltrating CD8+ T cells showed higher expression of bothIL-15Ra and cc chain than CD4+ T cells, which may explain the improve-ment of CD8+ T-cell growth in the presence of IL-2 + IL-7 + IL-15.Immunohistochemical identification of IL-15 and the higher mRNA expres-sion of IL-15Ra, IL-7 and cc chain in CCC heart tissues compared withcontrol individuals indicate in situ production of survival cytokines andtheir receptors in CCC hearts. Together, our results suggest that local pro-duction of IL-7 and IL-15 may be associated with the maintenance andpredominance of CD8+ T cells, the cells effecting tissue damage in CCChearts.

doi: 10.1111/j.1365-3083.2007.01987.x..................................................................................................................................................................

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362 Journal compilation � 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 362–371

gene expression levels of IFN-c-inducible chemokinesMCP-1, IP-10 and MIG, as well as the chemokine recep-tors CCR2 and CXCR3, were selectively upregulated inCCC heart tissue [7]. The heart infiltrate in CCC shows a2:1 ratio of CD8+ over CD4+ T cells [2, 3, 5] and granzymeA is detected in the heart lesions of CCC patients, suggest-ing a role for cytotoxicity in heart tissue damage [8]. It hasbeen observed that CD8+ T cells accumulate in the scarceareas of heart tissue where T. cruzi is found [9]. Recently,we observed that CD8+ T cells derived from a heart-infil-trating T-cell line from a CCC patient were specific toT. cruzi epitopes [10]. The factors leading to the accumula-tion of CD8+ T cells in CCC heart tissue are still unknown.It can be hypothesized that survival cytokines, such asIL-2, IL-7 and IL-15, play a role in this phenomenon.

Interleukin-2, IL-15 and IL-7, are known to have effectson CD4+ and CD8+ T-cell development, survival and func-tion. IL-15, IL-7 and IL-2 bind to multimeric receptorsthat share the common c chain (cc) subunit (CD132). Eachcytokine, however, interacts with a unique, ligand-specifica-chain receptor. IL-2 and IL-15 receptors also share ab chain (CD122) and distinct associations of cytokinereceptor chains generate receptors with high or intermedi-ate affinity for each cytokine [11]. Even though IL-15 andIL-2 share several properties, it is known that IL-2 and IL-15 can have very distinct roles in T-cell biology [12, 13].IL-2 promotes T-cell activation and proliferation, and sig-nalling through the IL-2 receptor (IL-2R) complex mayalso trigger the death or suppression of activated lympho-cytes [14–16]. On the other hand, signalling through theIL-15R complex is necessary for the maintenance of mem-ory CD8+ T cells and for NK-cell development [17, 18].IL-15 also exists as a membrane-associated cytokine andthe membrane IL-15 ⁄ IL-15Ra can be internalized andrecycled back to cell surface promoting longer IL-15 activ-ity. This process may be involved in the long-term CD8+

memory T-cell survival [19]. It has been shown that themaintenance of CD8+ naıve T cells require T-cell receptor(TCR) signalling through MHC and IL-7, whereas CD8+

memory T cells require IL-15 or IL-7 [20–23].Considering the role of IL-2, IL-7 and IL-15 in the

growth and maintenance of CD8+ T cells, in the presentstudy we investigated the involvement of such cytokinesand their receptors in the accumulation of CD8+ T cellsin CCC heart lesions.

Materials and methods

PBMC and PBMC-derived T-cell lines. Peripheral bloodsamples of Chagas disease patients with CCC were collec-ted with anticoagulant (heparin or EDTA) for this study.Sample collection procedures have been approved by theInternal Review Board. Mononuclear cells from hepari-nized peripheral blood samples were obtained by Ficollgradient centrifugation. Thawed PBMC were resuspended

in DMEM medium with 10% of the human serum.Viability was assessed and generally exceeded 90%. TheT-cell line from a CCC patient (P#12) was obtained fromPBMC stimulated with phytohaemagglutinin (PHA)(5 lg ⁄ ml) and 106 feeder cells (50-Gy irradiated PBMC)in 1 ml of Dulbecco’s modified Eagle’s medium supple-mented with 2 mM of L-glutamine, 1 mM sodium pyru-vate, MEM’s non-essential amino acids and MEM’svitamins (all from GIBCO, Grand Island, NY, USA),50 lg ⁄ ml of gentamicin, 10 mM HEPES buffer, 10% ofnormal human serum (complete medium) plus 40 UI ⁄ mlhuman recombinant interleukin-2 (Proleukin IL-2;Chiron, Emeryville, CA, USA) in a humidified, 5% ofCO2 atmosphere. After two rounds of expansion (10–20 days), lymphoblasts were re-stimulated and culturedin DMEM 10% human serum supplemented with IL-2(100 UI ⁄ ml), IL-7 (5 ng ⁄ ml; PeproTech, Rocky Hill,NJ, USA) and IL-15 (5 ng ⁄ ml; PeproTech) and stimula-ted by PHA (Sigma, St Louis, MO, USA). CD8+ cellswere purified using magnetic beads attached to mono-clonal antibody against human CD8 (M450; Dynal, Oslo,Norway). The beads were removed using Detach-a-Beadantibodies (Dynal). The resulting CD8+ T-cell line had94% of CD8+ cells and 0.3% of CD4+ cells, the CD8),pass-through T-cell line had 90.4% of CD4+ and 1.2%of CD8+ cells and was referred as a CD4+ T-cell line.

Heart-infiltrating T-cell lines. Right ventricle endomyo-cardial biopsy fragments from 11 CCC patients wereminced and cultured in 96-well flat-bottom cultureplates in DMEM (Gibco, Grand Island, NY, USA) with10% of inactivated human serum, supplemented with40 U ⁄ ml IL-2 (Hoffman-La Roche, Nutley, NJ, USA), inthe presence of autologous irradiated (50 Gy) PBMC(105 ⁄ well) as described [4]. Lymphoblasts were laterexpanded by two 15-day rounds of re-stimulation withPHA (2.5 lg ⁄ ml), 50-Gy irradiated PBMC (106 ⁄ ml) incomplete medium plus 40 U ⁄ ml of IL-2. After two tothree rounds of expansion, lymphoblasts were cultured inDMEM, 10% human serum supplemented by IL-2(100 U ⁄ ml), IL-7 (5 ng ⁄ ml) and IL-15 (5 ng ⁄ ml) and sti-mulated by PHA. No Trypanosoma cruzi parasite growthwas observed during endomyocardial biopsy explant cul-tures, as detected by direct visualization of the highlymobile trypomastigote forms.

Immunohistochemistry for the detection of IL-15 in the heartlesion. Fragments of myocardium of 15 CCC patients andof four control individuals, without inflammatory cardiacdisease, were frozen in isopentane and maintained in liquidnitrogen. Sections (5 lm) of frozen myocardial fragmentswere fixed in acetone and incubated with mouse anti-CD68primary antibody (1:200) from Dako-Patts (Glostrup,Denmark) to define macrophages or mouse anti-IL-15(1:100) from R&D. The secondary antibody was biotinyl-ated rabbit anti-mouse IgG (Dako, Ely, UK), diluted to1:400 and incubated during 30 min at 37 �C. The reaction

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product was detected with avidin–biotin–peroxidase com-plex according to ABC kit protocol (Vector Laboratories,Burlingame, CA, USA), and the reaction was developedwith diamino-benzidine (Sigma). The negative control wasassessed by elimination of primary antibody. The slideswere counterstained with haematoxylin and eosin. CD68+

for macrophages or IL-15+ mononuclear cells were countedin 25 fields, 400· optical magnification. In the standard-ization of the anti-human IL-15 antibody, we utilizedmononuclear cells of peripheral blood adhered in slidesand stimulated with lipopolysaccharide (LPS), 10 lg ⁄ ml,during 48 h.

Flow cytometry assays. The surface phenotype of lympho-blasts was identified after immunostaining with monoclo-nal antibodies directed to the CD3, CD4, CD8, TCRab,TCRcd, CD56, CD16 surface markers (BD Biosciences,San Jose, CA, USA) labelled with fluorochromes and flowcytometric analysis (FACScan; Becton-Dickinson, SanJose, CA, USA). The phycoerythrin-conjugated (PE) anti-bodies against CD25 (IL-2Ra), CD122 (IL-2Rb), CD132(IL-2Rc) were all purchased from BD PharMingen (SanDiego, CA, USA). For IL-15Ra-chain staining, cells wereincubated with an IL-15 mutant ⁄ Fcc2a fusion that bindsto the a-chain [24], followed by staining with biotinylat-ed goat anti-mouse IgG antibody. Cells were then washedand stained with PE-streptoavidin. Twenty thousandevents were collected and analysed on a FACScan flow cy-tometer and CellQuest software (Becton-Dickinson; Bio-sciences). Based on the characteristics of the forward andside-scatter patterns, a gate was set on the lymphocytesand the expression of IL-2R and IL-15R chains was fol-lowed separately on CD3+CD4+ and CD3+CD8+ cells.

Assessment of in vitro growth. T cells from long-termPBMC or heart-infiltrating T-cell lines from CCCpatients (2 · 105 cells ⁄ well) were cultured with 1 · 106

irradiated PBMC plus PHA (2.5 lg ⁄ ml) in the presenceof different cytokines: IL-2 (100 IU ⁄ ml), IL-7 and IL-15(10 ng ⁄ ml) or their combination. Fresh medium supple-mented with cytokines was added every 3 days. To deter-mine the survival and expansion of T cells, every2–3 days from day 6, the numbers of viable cells wereestimated using trypan blue exclusion, and the cells werere-suspended in fresh medium containing the correspond-ing cytokines.

Long-term effects of cytokines on heart-infiltrating CD8+

T-cell growth. T-cell lines were established from endomyo-cardial biopsy of CCC patients and cultured in the presenceof human recombinant IL-2 (100 lg ⁄ ml). After two tothree stimuli with PHA (2.5 lg ⁄ ml), the lines were cul-tured in the presence of IL-2 + IL-7 + IL-15. The pheno-type of T-cell line was monitored after two or more stimuliwith PHA in the presence of IL-2 + IL-7 + IL-15.

Real-time reverse transcriptase-PCR analysis. RNA sam-ples for the real-time reverse transcriptase (RT)-PCRexperiments came from explanted left ventricular free wall

heart samples obtained at transplantation from 11 CCC(serological diagnosis and positive epidemiology) end-stageheart failure patients and three non-failing donor heartsfrom normal adult heart tissue not used for cardiac trans-plantation. The total RNA was extracted using the Trizol�

method (Life Technologies Inc., Grand Island, NY, USA).The integrity of the RNA was confirmed by gel electro-phoresis. For cDNA synthesis, 5 lg of total RNA, 1 lloligodT (500 lg ⁄ ml), 1 ll dNTP (10 mM of dATP,dCTP, dGTP and dTTP), DEPC water q.s.p. 12 ll, wereincubated for 5 min at 40 �C in a 200-ll tube. Later, 4 llTranscription Buffer 5x (Tris-HCl 250 mM pH 8.3, KCl375 mM, MgCl2 15 mM), 2 ll DTT 0.1 M, 1 ll inhibitorRNase (40 U ⁄ ll) (RNAse OUT�; Invitrogen, Carlsbad,CA, USA) and 1 ll Super-script II� Reverse Transcriptase(200 U ⁄ ll) (Invitrogen, Carlsbad, CA, USA) were added;the reaction was heated for 50 min at 42�C and 15 min at70�C. We designed forward and reverse primers for quanti-tative real-time RT-PCR using Primer Express (PEApplied Biosystems, Foster City, CA, USA), for amplifica-tion of the desired gene with the following primersequences: IL-2 (Acc. no. V00564) sense 5¢-GACCCAGG-GACTTAATCAGCAAT-3¢, anti-sense 5¢-CTGTCTCATC-AGCATATTCACACATG-3¢; IL-2Ra (Acc. no. X01057)sense 5¢-GCGTCTGCAAAATGACCCAC-3¢, anti-sense5¢-CTGAGGCTTCTCTTCACCTGGA-3¢; IL-2Rb (Acc.no. M26062) sense 5¢-ATCAACTGCAGGAACACCGG-3¢, anti-sense 5¢-CTGGGAAAAGAACTTCGAGGG-3¢;IL-2Rc (Acc. no. D11086) sense 5¢-TCTGGCTGGAACG-GACGAT-3¢, anti-sense 5¢-ACACCACTCCAGGCCG-AAA-3¢; IL-7 (Acc. no. J04156) sense 5¢-TGAACTGCACTGGCCAGGTTAA-3¢, anti-sense 5¢-CTTCCAAACTCT-TTGTTGGTTGGG-3¢; IL-7Ra (Acc. no. M29696) sense5¢-GATGAAAACAAATGGACGCATGT-3¢, anti-sense5¢-GTGATCAGGGATGGATCGAACTT-3¢; IL-15 (Acc.no. U14407) sense 5¢-GGTTCACCCCAGTTGCAAAG-TAAC-3¢, anti-sense 5¢-TATCATGAATACTTGCATCT-CCGGA-3¢; IL-15Ra (Acc. no. U31628) sense 5¢-TGAGCGCTGTGTCTCTCCTG-3¢, anti-sense 5¢-CCATGGCTTCCATTTCAACG -3¢; glyceraldehyde-3-phosphate dehy-drogenase (GAPDH; Acc. no. M33197) sense 5¢-TGGT-CTCCTCTGACTTCAACA-3¢, anti-sense 5¢-AGCCAAATTCGTTGTCATACC-3¢. Real-time PCR reactions werecarried out in an ABI Prism 7500 Sequence Detection sys-tem (PE Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using the SYBR Green PCRMaster Mix (PE Applied Biosystems), according to themanufacturer’s instructions, using 5 ll of diluted cDNAas a template. All the samples were tested in triplicate withthe reference gene GAPDH, a housekeeping gene for nor-malization of data. For all genes we constructed standardcurves and determined the slope to calculate the PCR effi-ciency according to Pfaffl [25]. Normalization and foldchange were calculated with the DDCt method withGAPDH as the reference mRNA [26].

364 IL-15 and IL-7 in Chagas’ Cardiomyopathy S. G. Fonseca et al...................................................................................................................................................................

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Statistical analysis. The non-parametric Mann–Whitneyrank sum test was used to compare the numbers ofIL-15+ and CD68+ cells in heart tissues from CCCpatients and non-inflammatory heart disease samples.

Results

Phenotypic characterization of derived heart-infiltrating

T-cell lines from CCC patients grown on IL-2

To investigate the expansion of heart-infiltrating T cellsby T-cell trophic cytokines, heart-infiltrating T-cell lineswere established culturing small fragments of heart tissuebiopsies from 10 CCC patients in the presence of IL-2and autologous PBMC-irradiated feeder cells. The average±standard deviation of the percentage of CD3+ T cellsamong these T-cell lines was 94.0 ± 5.1% and an over-whelming majority was TCRab+ (99.0 ± 0.4%). Lessthan 1% of cells were TCRcd+ or CD56+CD16+ (NKcells). A predominance of 6:1 of CD4+ (74.6 ± 11.0%)over CD8+ T cells (11.3 ± 10.4%) was observed. This isin sharp contrast to the 0.5:1 CD4:CD8 T-cell ratiosfound on direct immunohistochemistry analysis of CCCendomyocardial biopsies [2, 3].

Effect of IL-7 and IL-15 on the expansion of CD8+ and

CD4+ T cells from PBMC-derived T-cell line from a CCC

patient

We thus compared the effect of IL-15 and IL-7 withIL-2 on the expansion of CD4+ and CD8+ T cells fromT-cell lines obtained from a CCC patient. For that mat-ter, we used isolated CD8+ and CD4+ T cells, obtained

from a PBMC-derived T-cell line from a CCC patient.The CD8+ and CD4+ T-cell lines were stimulated withPHA and irradiated feeder cells plus IL-2, IL-7, IL-15alone or in combination, and the cell growth was fol-lowed for 31 days. Fresh culture medium with cytokineswas added twice a week. Figure 1 shows that in thepresence of IL-2 alone, the number of viable CD4+ Tcells was stable until 31 days after stimulation withPBMC-irradiated cells plus PHA. In contrast, thegrowth of the CD8+ T-cell line was clearly reduced15 days after stimulus, in comparison to CD4+ T cellsunder IL-2 alone (Fig. 1). Both CD4+ and CD8+ Tcells show increased expansion under IL-7 and IL-15,when compared with IL-2 alone; while the combinationof IL-2, IL-7 and IL-15 increased both expansion andmaintenance of viability over time.

Expression of chains of IL-2R and cc chain on PBMC-derived

CD8+ and CD4+ T lymphocytes

We investigated whether the failure of IL-2 to inducethe growth of long-term PBMC-derived CD8+ T cellsfrom a CCC patient was dependent on the differentialexpression of IL-2R chains. For that matter, we evaluatedthe kinetics of the expression of IL-2Ra (CD25),IL-2 ⁄ IL-15Rb (CD122) and cc chain (CD132) on thePBMC-derived CD8+ and CD4+ T-cell lines culturedwith IL-2, 15 days after the last stimulation with PHAand irradiated feeder cells. We observed that the medianof fluorescence intensities for IL-2Ra and cc chain weresubstantially higher on CD4+ T cells than on CD8+ Tcells (Fig. 2A). This pattern did not change at day 10after PHA stimulation (data not shown). Similarly, we

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Figure 1 Growth of CD8+ and CD4+ T cells

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of cytokines. CD8+ and CD4+ T-cell lines

(2 · 105) isolated from a PBMC-derived

T-cell line from a Chagas disease patient were

cultured with 106 irradiated allogeneic

PBMC as feeder cells and PHA (2.5 lg ⁄ ml)

in presence of IL-2 (100 IU ⁄ ml), IL-7

(5 ng ⁄ ml) and IL-15 (5 ng ⁄ ml) or with a

combination of the three cytokines. Viable

cells were counted in a Neubauer chamber

from day 6 to day 31. Each time-point repre-

sents averages of cell numbers from three dif-

ferent experiments.

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also observed that the percentages of IL-2Ra- and cc

chain-positive cells were much lower on CD8+ T cellsthan on CD4+ T cells from the PBMC-derived T-cell linefrom the CCC patient (Fig. 2B).

Effect of IL-2, IL-7 and IL-15 on the expansion of

heart-infiltrating T-cell lines from Chagas disease patients

After the observation that IL-15, IL-2 and IL-7 wereimportant for the long-term growth of peripheral CD8+

T-cell lines, we investigated the effect of the combinationof IL-2 + IL-7 + IL-15 on the expansion of heart-infiltra-ting T-cell lines from CCC patients. Figure 3 shows thegrowth curves of distinct heart-infiltrating T-cell linesfrom five CCC patients, after PHA and irradiated feederstimuli. Although there was some interindividual vari-ation of expansion levels, the combination of IL-2, IL-7and IL-15 promoted an increase in the number of viableT cells in comparison to IL-2 alone, visible on day 12after stimulus in all T-cell lines.

Differential expansion of CD8+ T cells from heart-infiltrating

T-cell lines from CCC patients in the presence of IL-2, IL-15

and IL-7

To address whether IL-15 and IL-7 were related with themaintenance of CD8+ T cells in the heart infiltrate of Cha-gas disease patients, we analysed the long-term growth ofheart-infiltrating T-cell lines from Chagas disease patients

in the presence of IL-2, IL-7 and IL-15. These heart-infil-trating T-cell lines were established initially with IL-2and autologous-irradiated feeder cells, and the average pro-portion of CD8+ T cells was 11.34%, ranging from 0.4%to 38%. The effect of long-term culture with IL-2, IL-7and IL-15 on the expansion of CD8+ T cells from heart-infiltrating T-cell lines from CCC patients is shown inFigure 4. Half of the heart-derived T-cell lines showed anincrease in the proportion of CD8+ T cells (Fig. 4; CD8+

T cells after expansion ⁄ CD8+ T cells before expansion ran-ging from 2 to 72).

In situ expression of IL-15 on heart tissue from CCC patients

Considering the importance of IL-15 in the growth andmaintenance of CD8+ T cells from heart-infiltrating T-celllines and the predominance of CD8+ T over CD4+ T cellin the heart infiltrate of Chagas disease patients, we nextinvestigated the expression of IL-15 in the endomyocardialbiopsies of 14 CCC patients. As IL-15 can be secreted bymacrophages, the number of CD68+ macrophages was alsoquantified in the same heart tissues. Immunohistochemis-try analysis showed that the cell infiltrates from Chagasdisease patients have a significantly higher numbers of bothCD68+ cells and IL-15+ cells when compared with thehearts from control donors (Fig. 5). The average number ofIL-15+ cells ⁄ 400· field among CCC patients was 7.7 ±10.1, compared with 0.48 ± 0.4 cells among control sam-ples, a 16-fold difference (P = 0.01); the average number

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Figure 2 Expression of IL-2 receptor chains on CD8+ and CD4+ T cells from PBMC-derived T-cell lines from a Chagas disease patient, cultured in

presence of IL-2. Cells from CD8+ and CD4+ T-cell lines (2 · 105) isolated from PBMC-derived T-cell line from a Chagas disease patient were cul-

tured with 106 irradiated allogeneic feeder cells and PHA (2.5 lg ⁄ ml) in presence of IL-2 (100 IU ⁄ ml). (A) The relative expression of IL-2Ra,

IL-2 ⁄ IL-15Rb and cc on CD8 (shaded histogram) and on CD4 (solid line) T-cell lines were measured on day 0 by double stained with FITC or

PE-labelled antibodies to IL-2R chains and CD8 and CD4 detected by flow cytometry. (B) The percentage of positive cells to IL-2Ra, IL-2 ⁄ IL-15Rband cc in CD8+ and CD4+ T-cell lines were measured on day 0 and 10 after PHA stimulus and are represented by columns in the graph.

366 IL-15 and IL-7 in Chagas’ Cardiomyopathy S. G. Fonseca et al...................................................................................................................................................................

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of CD68+ cells ⁄ 400· field among CCC patients was9.3 ± 9.1, compared with 2.2 ± 0.8 among control sam-ples, a fourfold difference (P = 0.01) (Fig. 5E). The ratio ofIL-15+ ⁄ CD68+ cells among heart samples of Chagas dis-ease patients was fourfold higher compared with controlsamples (Fig. 5F; P = 0.02), suggesting an increasedactivation state.

Expression of IL-2, IL-7, IL-15 and their receptors on

intralesional T-cell lines and in the heart tissue from

CCC patients

Considering that heart-infiltrating T-cell lines expandedafter long-term culture with a combination of IL-2 + IL-7 + IL-15, we investigated the expression of IL-2 andIL-15 receptor chains in heart-derived T-cell lines. Weevaluated the surface expression of IL-2Ra, IL-15Ra andIL-2 ⁄ IL-15Rb and cc chain in CD8+ and CD4+ T cellsfrom heart-infiltrating T-cell lines from three CCCpatients, cultured with a combination of IL-2, IL-7 and IL-15. At day 15 post-stimulus, the proportion of IL-15Ra+

and IL-2 ⁄ IL-15Rb+ cells was higher on the CD8+ T cellsin all three heart-infiltrating T-cell lines than on CD4+ Tcells (Fig. 6). However, no difference was detected in theexpression of IL-2Ra and cc chain on the CD8+ and CD4+

T cells in the same heart-infiltrating T-cell lines culturedin the same conditions (Fig. 6). In addition, the median offluorescence intensity of IL-15Ra was higher on CD8+ Tcells (9.5, 62.3 and 155.3 respectively) than on CD4+ Tcells (3.4, 3.2 and 5.42 respectively).

To assess whether IL-2, IL-7 and IL-15 and theirreceptors are also expressed in CCC heart tissue, we usedreal-time PCR in heart tissue samples from 11 CCCpatients in comparison to heart tissues from three non-inflammatory heart diseases. Figure 7 shows that theexpression average of mRNA levels of genes IL-15Ra,

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the growth of heart-infiltrating T-cell lines.

After three to five stimuli with 106 irradiated

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were washed and split into two different cul-

ture conditions, one with IL-2 (100 IU ⁄ ml)

and the other with IL-2 + IL-7 + IL-15

(100 IU ⁄ ml, 5 ng ⁄ ml and 5 ng ⁄ ml respect-

ively) in presence of PHA and feeder cells,

counted every 3 days.

0

10

20

30

40

P#01

P#02

P#03

P#04

P#05

P#06

80

90

100

IL-2 IL-2+7+15

2-3 stimuli 2-5 stimuli

P#07

P#08

P#09

P#10

% C

D8+

Figure 4 Chronic exposure to IL-2 + IL-7 + IL-15 rescues the growth

of CD8+ T cells from heart-infiltrating T-cell lines. Heart-derived T-cell

lines were initially obtained from heart biopsies from 10 CCC patients

were initially stimulated with 106 irradiated autologous PBMC plus

PHA (2.5 lg ⁄ ml) and IL-2 (100 IU ⁄ ml). After three to five stimuli,

the T-cell lines were re-stimulated with IL-2 + IL-7 + IL-15

(100 IU ⁄ ml, 5 ng ⁄ ml and 5 ng ⁄ ml respectively) in presence of PHA

and feeder cells and the percentile of CD8+ T cells was measured by

flow cytometry.

S. G. Fonseca et al. IL-15 and IL-7 in Chagas’ Cardiomyopathy 367..................................................................................................................................................................

� 2007 The Authors

Journal compilation � 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 362–371

IL-7 and IL-2Rc in CCC myocardium was 2.7- toninefold higher in the heart of CCC patients than in thehearts of normal donors, suggesting that IL-7, IL-15Ra,IL-2Rc may also be upregulated among infiltrating Tcells in CCC. As the expression of IL-2, IL-2 ⁄ IL-15Rband IL-7Ra RNA was undetectable in heart tissue from

control individuals, the relative expression of these recep-tors and cytokines could not be addressed. However, theaverage Ct values of IL-2, IL-2 ⁄ IL-15Rb and IL-7Ra inheart tissue from the CCC patients were 31.9, 27.1 and27, respectively, indicating that these genes were alsoexpressed in CCC heart tissue.

0

10

20

30

40

CD68

p = 0.01 p = 0.01

Mea

n n

um

ber

po

siti

vece

lls/f

ield

400

x

0.0

0.5

1.0

1.5

2.0

CCC CN

p = 0.02

IL-1

5+/C

D68

+ fi

eld

400

X

E

C D

A B

F

CNCCCCNCCC

IL-15

Figure 5 Expression of IL-15 and CD68 in

the heart tissues of Chronic Chagas disease

patients and control individuals. Heart biop-

sies from CCC patients and controls were pre-

pared for immunohistochemical analysis, and

the presence of IL-15 and CD68 was evaluated

by the immunoperoxidase method. The pres-

ence of IL-15 and CD68+ macrophages was

revealed by using diaminobenzidine tetrahy-

drochloride as the substratum for the peroxi-

dase, which generated a brown colour. Results

are representative of heart fragments from 14

CCC and four control individuals. CD68+

cells in one control individual (A) and in one

CCC patient (B). IL-15 in one control individ-

ual (C) and in one CCC patient (D). (A and C)

Haematoxylin; (B and D) DAB and Haema-

toxylin. (A–D) 400·. (E) The number of IL-

15+ and CD68+ cells was detected in the heart

tissue biopsies. (F) The ratio of IL-15+

cells ⁄ CD68+ cells among heart biopsies of

CCC patients and control samples. Horizontal

bars represent mean values.

0

20

40

60

80

100 CD8CD4

%R

2-LI

αsll

ec

+

P#07 P#01 P#09

P#07 P#01 P#09

P#07 P#01 P#09

P#07 P#01 P#09

0

20

40

60

80

100

%R

51-

LIα

sl le

c +

0

20

40

60

80

100

%R

51-

LI/2-

LIβ

slle

c +

0

20

40

60

80

100

%γ c

slle

c +

Figure 6 Expression of IL-2 ⁄ IL-15 receptor

chains on CD8+ and CD4+ T cells from

heart-derived T-cell lines from CCC patients,

cultured in presence of IL-2 + IL-7 + IL-15.

Cells from heart-derived T lines (2 · 105)

from three distinct Chagas disease patients

were cultured with 106 irradiated allogeneic

feeder cells and PHA (2.5 lg ⁄ ml) in presence

of IL-2 (100 IU ⁄ ml) and IL-2 + IL-7 + IL-

15 (100 IU ⁄ ml, 5 ng ⁄ ml and 5 ng ⁄ ml

respectively). At 15 days after PHA stimulus,

cells were stained with FITC- or PE-labelled

antibodies against IL-15Ra, IL-2Ra, IL-2 ⁄ IL-

15Rb, c chain, anti-CD8 and anti-CD4. The

expression of receptor chains was detected by

flow cytometry. The bars represent the per-

centages of CD8+ and CD4+ cells expressing

the receptor chains.

368 IL-15 and IL-7 in Chagas’ Cardiomyopathy S. G. Fonseca et al...................................................................................................................................................................

� 2007 The Authors

Journal compilation � 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 362–371

Discussion

In the present work, we showed that the combination ofIL-15, IL-7 and IL-2 promoted the in vitro expansion ofCD8+ T cells from both PBMC-derived and heart-infil-trating T-cell lines from CCC patients. Such expansionmight be dependent on the selective expression ofIL-15Ra, and cc chain on CD8+ T cells from heart-infil-trating T-cell lines. Accordingly, higher heart tissueexpression of IL-15Ra, IL-15, and IL-7 was observed inCCC patients. Our results are consistent with the ideathat the accumulation of CD8+ T cells in the heart infil-trate of CCC patients may be dependent on the local pro-duction of IL-15 and IL-7.

The finding that IL-7 and ⁄ or IL-15 can rescue theproliferation and maintenance of the viability of IL-2-cul-tured CD8+ T cells from a PBMC-derived T-cell linefrom a CCC patient suggests that they play a major rolein the in vitro growth of CD8+ T cells (Fig. 1). In accord-ance with our results, it has been shown by others thatIL-7 [27, 28] and IL-15 [29] preferentially expand theCD8+ T-cell population. The mechanisms by whichIL-15 may contribute to the maintenance of memoryT cells are not fully understood. It has been shown thatthe cell surface IL-15Ra ⁄ IL-15 complex can undergoendosomal internalization, survive lysosomal degradationand recycle to the cell surface [19], contributing to thelong-term survival of CD8+ memory T cells. In our sys-tem, while a proliferative effect of IL-7, IL-15 or theircombination with IL-2 was obvious in the first 2 weeks,until the peak of growth, we cannot ascertain whetherincreased proliferation, inhibition of apoptosis or bothwere operative during the last 2 weeks of the culture,where the cell numbers were stable or decreased. The find-ing that CD8+ T cells from a PBMC-derived T-cell line

from a CCC patient express less IL-2Ra and cc thanCD4+ T cells (Fig. 2) could partially explain the relativerefractoriness of CD8+ T cells to IL-2 (Fig. 1), in linewith the published findings [30–32]. Moreover, the find-ing that CD4+ T cells from the PBMC-derived T-cell linealso increased in numbers after incubation with IL-7,IL-15 or IL-2 + IL-7 + IL-15 in comparison to IL-2 alone(Fig. 2) is also in line with the published literature [33].

The combination of IL-2 + IL-7 + IL-15 also preferen-tially expanded CD8+ T lymphocytes from intralesionalT-cell lines (Fig. 4). In addition, we have also shown thatCD8+ T lymphocytes from the three distinct intralesionalT-cell lines present higher expression of the IL-15Ra andIL-2 ⁄ IL-15Rb, when compared with CD4+ T lympho-cytes (Fig. 5) as observed by others [19, 30]. This is con-sistent with the selective in vitro growth of the heart-infiltrating CD8+ T lymphocytes in the presence of IL-2 +IL-7 + IL-15 by a receptor-driven mechanism. The mech-anism of increased responsiveness to IL-15 observedamong heart-infiltrating CD8+ T cells (increased IL-15Raand cc) seems thus to be distinct from that ofPBMC-derived CD8+ T cells, which showed reducedIL-2Ra and cc expression instead (Fig. 2). We cannotexclude that the refractoriness to IL-2 observed amongheart-infiltrating T cells could be due to distinct post-transductional signalling of IL-2.

The finding of increased mRNA expression ofIL-15Ra and cc in the CD8+ T-cell rich CCC myocardialtissues (Fig. 7) is in line with the surface expression dataon heart-infiltrating T-cell lines (Fig. 6), suggesting thatin situ T cells effectively express such receptors in vivo.The observation of increased levels of IL-7 mRNA(Fig. 7) and an increased number of IL-15+ cells, in thehearts from CCC patients (Fig. 5), indicates that bothIL-7 and IL-15, ligands for the above-mentioned recep-tors, are indeed expressed in the CCC heart tissue. Thefinding that IL-15 mRNA was expressed at similar levelsin control and the CCC heart tissue was in conflict withthe increased expression of immunologically detectableIL-15 protein in the CCC heart tissue. Nevertheless, ithas been reported that IL-15 mRNA is constitutivelyexpressed in many tissues, and its regulation is mainlypost-transcriptional [19, 34]. Together, our results areconsistent with locally produced IL-7 and IL-15 beingmajor growth factors for CD8+ T lymphocytes in CCCheart tissue.

Interleukin-15 has also been identified as an import-ant mediator in several chronic inflammatory diseases[34–40]. IL-15 can synergize with the IL-12 for IFN-cproduction [41], and IL-15 synergizes with IL-7 toenhance the ability of antigen-specific CD8+ and CD4+

T cells to produce IFN-c [42]. Moreover, IFN-c itselfinduces the expression of IL-15 [34]. The finding thatthe ratio of IL-15+ ⁄ CD68+ cells is increased in CCCheart tissue suggests that more CD68+ macrophages are

0.1

1

10

100F

old

ch

ang

e (v

s. n

orm

al h

eart

)

IL-15 IL-15Rα IL-7 IL-2Rα IL-2Rγ

Figure 7 Real-time RT-PCR analysis of expression of mRNA levels of

genes IL-15, IL-15Ra, IL-7, IL-2Ra and IL-2Rc in CCC myocardium.

Normalization and fold change were calculated with the DDCt method,

the data are presented as fold change in gene expression normalized to

the housekeeping gene GAPDH and relative to the three normal hearts.

Data represent mean of triplicate samples obtained from 11 CCC myo-

cardium samples. The horizontal bar stands for the median. Genes

whose median fold-change was above 2 or below 0.5 of control tissues

were considered differentially expressed.

S. G. Fonseca et al. IL-15 and IL-7 in Chagas’ Cardiomyopathy 369..................................................................................................................................................................

� 2007 The Authors

Journal compilation � 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 362–371

induced to produce IL-15, possibly due to the inflamma-tory milieu. It is thus likely that the locally expressedIL-15, possibly produced by CD68+ macrophages, maycontribute to the increased production of proinflammato-ry cytokines, such as IFN-c and TNF-a, observed inCCC heart tissue [4, 5].

Taken together, our results suggest that the accumula-tion of CD8+ T cells in the heart tissue of CCC patientsmay be due, at least in part, to the local expression ofsurvival cytokines, possibly induced by IFN-c. IL-7 andIL-15 which may act on CD8+ cells bearing specific cyto-kine receptors, allowing their selective growth, survivaland maintenance. Their ongoing production of IFN-ccould in turn upregulate local production of IL-15, in apositive feedback loop. Furthermore, this mechanismwould lead to the maintenance of Th1-type inflammatorycells in heart tissue. Whether such granzyme+CD8+ Tcells have a role in heart tissue damage in CCC, or aremerely a collection of exhausted T cells remains to beestablished.

Acknowledgments

We thank Ms Priscila Teixeira for helping in heart tissuecollection. This work was supported by grants from FAP-ESP nº 97 ⁄ 13431-5 (to S.G.F.), CNPq nº 42.0006 ⁄ 99-0and nº 520533 ⁄ 97-6 (to E.CN).

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S. G. Fonseca et al. IL-15 and IL-7 in Chagas’ Cardiomyopathy 371..................................................................................................................................................................

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Journal compilation � 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 66, 362–371