Scholarly article on topic 'Presence of Antigen-Experienced T Cells with Low Grade of Differentiation and Proliferative Potential in Chronic Chagas Disease Myocarditis'

Presence of Antigen-Experienced T Cells with Low Grade of Differentiation and Proliferative Potential in Chronic Chagas Disease Myocarditis Academic research paper on "Biological sciences"

CC BY
0
0
Share paper
Academic journal
PLoS Negl Trop Dis
OECD Field of science
Keywords
{""}

Similar topics of scientific paper in Biological sciences , author of scholarly article — Rafael J. Argüello, Carlos Vigliano, Patricia Cabeza-Meckert, Rodolfo Viotti, Fernando Garelli, et al.

Academic research paper on topic "Presence of Antigen-Experienced T Cells with Low Grade of Differentiation and Proliferative Potential in Chronic Chagas Disease Myocarditis"

OPEN 3 ACCESS Freely available online (pj»PLOS | tropical diseases

Presence of Antigen-Experienced T Cells with Low Grade of Differentiation and Proliferative Potential in Chronic Chagas Disease Myocarditis

Rafael J. Argüello1®, Carlos Vigliano29, Patricia Cabeza-Meckert2, Rodolfo Viotti3, Fernando Garelli4, Liliana E. Favaloro5, Roberto R. Favaloro5, Rüben Laguens2, Susana A. Laucella1'3*

1 instituto Nacional de Parasitología ''Dr. Mario Fatala Chabén", Buenos Aires, Argentina, 2 Departamento de Patología, Hospital Universitario Fundación Favaloro, Buenos Aires, Argentina, 3 Servicio de Cardiología, Seccion de Chagas, Hospital interzonal General de Agudos ''Eva Peron'', San Martín, Buenos Aires, Argentina, 4 Laboratorio de Eco-Epidemiología de la enfermedad de Chagas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina, 5 Departamento de Trasplante Intratoracico. Hospital Universitario Fundacion Favaloro, Buenos Aires, Argentina

Abstract

Background: The main consequence of chronic Trypanosoma cruzi infection is the development of myocarditis in approximately 20-30% of infected individuals but not until 10-20 years after the initial infection. We have previously shown that circulating interferon-y-secreting T cells responsive to Trypanosoma cruzi antigens in chronic Chagas disease patients display a low grade of differentiation and the frequency of these T lymphocytes decreases along with the severity of heart disease. This study thought to explore the expression of inhibitory receptors, transcription factors of type 1 or regulatory T cells, and markers of T cell differentiation, immunosenescence or active cell cycle in cardiac explants from patients with advanced Chagas disease myocarditis.

Methodology/Principal Findings: The expression of different markers for T and B cells as well as for macrophages was evaluated by immunohistochemistry and immunofluorescence techniques in cardiac explants from patients with advanced chronic Chagas disease submitted to heart transplantation. Most infiltrating cells displayed markers of antigen-experienced T cells (CD3+, CD4+, CD8+, CD45RO+) with a low grade of differentiation (CD27+, CD57", CD45RA", PD-1"). A skewed T helper1/T cytotoxic 1 profile was supported by the expression of T-bet;whereas FOXP3+ cells were scarce and located only in areas of severe myocarditis. In addition, a significant proliferative capacity of CD3+ T cells, assessed by Ki67 staining, was found.

Conclusions/Significance:The quality of T cell responses and immunoregulatory mechanisms might determine the pattern of the cellular response and the severity of disease in chronic Trypanosoma cruzi infection.

Citation: Arguello RJ, Vigliano C, Cabeza-Meckert P, Viotti R, Garelli F, et al. (2014) Presence of Antigen-Experienced T Cells with Low Grade of Differentiation and Proliferative Potential in Chronic Chagas Disease Myocarditis. PLoS Negl Trop Dis 8(8): e2989. doi:10.1371/journal.pntd.0002989

Editor: Mauricio Martins Rodrigues, Federal University of Sao Paulo, Brazil

Received October 11, 2013; Accepted May 19, 2014; Published August 21, 2014

Copyright: © 2014 Arguello et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the National Council for Scientific and Technical Research, Argentina (CONICET PIP №5686 to Susana A. Laucella). SAL is member of the Scientific Career, CONICET, Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Competing Interests: The authors have declared that no competing interests exist. * Email: slaucella@yahoo.com . These authors contributed equally to this work.

Introduction

The main consequence of chronic Trypanosoma cruzi (T. cruzi) infection is the development of myocarditis in approximately 2030% of infected individuals but not until 10-20 years after the initial infection [1]. Advanced chronic Chagas heart disease (cChHD) is characterized by dilated cavities with high degree of fibrosis and inflammation [2,3]. The analysis by immunohisto-chemical, but mainly by molecular techniques, of cardiac samples from chronically T. cruzi-infected subjects provided evidence of the association between parasite persistence and tissue damage in cChHD [2,4,5]. Reis et al. showed that inflammatory lesions were dominated by CD8+ lymphocytes, many of which expressed granzyme A [6]. Lymphocytes in these lesions express lymphocyte

function antigen-a (LFA-1), CD44, very late antigen-4 (VLA-4) [7] and cytotoxic lymphocyte antigen 4 (CTLA-4) [8]. A Th1 cytokine pattern predominated in the cardiac inflammatory cell infiltrate of Chagas disease patients with heart failure [9].Whereas some authors have shown increased peripheral levels of IFN-y in patients with severe heart disease [10-12], other studies have demonstrated an inverse association between disease severity and IFN-y production [8,13,14].We have previously shown that most IFN-y-secreting T cells in response to T. cruzi display a low grade of differentiation but high expression of the inhibitory receptor CTLA-4 in the circulation of subjects with chronic T. cruzi infection [8,15,16]. Conversely, the total T cell compartment in Chagas disease patients is enriched in highly differentiated T cells compared to uninfected controls [15-17].

Author Summary

Chagas disease is a neglected tropical disease affecting approximately 10 million people in the world. It is caused by infection with the protozoan Trypanosoma cruzi. As a consequence of migration flows, the disease has been also become established in non-endemic countries. In this study, the functional and phenotypic profile of inflammatory T cells were evaluated in heart tissues of patients with end-stage chronic Chagas disease by analyzing the expression of inhibitory receptors, transcription factors of type 1 or regulatory T cells, and markers of T cell differentiation, immunosenescence or active cell cycle. Most infiltrating cells displayed markers of antigen-experienced T cells with a low grade of differentiation and a significant proliferative capacity. A skewed T helper1/T cytotoxic 1 profile was supported by the expression of T-bet;whereas FOXP3+ cells were scarce. The quality of T cell responses and immunoregulatory mechanisms might determine the pattern of the cellular response and the severity of disease in Trypanosoma cruzi infection.

There is very limited data available on the degree of differentiation of T cells in heart lesions of cChHD, and a comprehensive analysis of the relationship of different T cell functions in Chagas disease myocarditis has not been performed. This study thought to explore the expression of inhibitory receptors, transcription factors of type 1 or regulatory T cells and markers of T cell differentiation, immunosenescence or active cell cycle in cardiac explants from patients with advanced cChHD submitted to heart transplantation.

Materials and Methods

Ethics statement

This study was approved by the Institutional Review Boards of the Hospital Universitario Fundación Favaloro (UIC (863) 1604), and all patients gave written informed consent for the heart transplant procedure.

Patients

Eight patients with severe cChHD (4 men and 4 women; mean age ± SD, 51.46 7.3 years) were admitted at Hospital Universitario Fundacion Favaloro in Buenos Aires, Argentina, during the period 1998-2008 to undergo orthotopic heart transplantation. Diagnosis of T. cruzi infection was confirmed when at least 2 out of 3 standard serological tests (enzyme-linked immunosorbent assay, indirect hemagglutination and immuno-fluorescence).were positive. Determination of cChHD was based on clinical, echocardiographic and electrocardiographic findings. Explanted hearts from patients with Giant cell myocarditis (GCM; n = 2) and idiopathic dilated cardiomyopathy (IDCM; n = 1) were also included as controls. Human lymph node and placental tissues from the Tissue Bank of the Pathology Lab were employed as positive staining controls. The cChHD patients included in this study had not received immunosup-pressive drugs, trypanocidal therapy or prophylactic benznida-zole by the time of this study.

Analysis of heart explants

Eight explanted hearts were weighed and fixed for 72 h in 10% phosphate-buffered formaldehyde. After fixation, transmural sections of the whole circumference of the left and right ventricle

at a plane equidistant from the base to the apex were collected and embedded in paraffin. A 5-mm-thick section from each region was stained with hematoxylin and eosin and Masson's trichrome solution. The interventricular septum of each heart was selected for histological and immunohistochemistry analysis. The diagnosis of myocarditis was defined according to the Dallas criteria taking into account the inflammatory infiltrate of the myocardium and the presence of necrosis and/or degeneration of adjacent myocytes [18]. The distribution of the inflammatory infiltrate was classified as focal, confluent or diffuse [19].

The median number of lymphocytes plus macrophages identified by the expression of CD3 and CD68, respectively, was calculated for 8 tissue samples from cChHD, 2 GCM samples and

1 IDCM sample assessed, as described in "Quantification of cells'' [20]. Myocarditis recorded in each tissue sample was considered as severe when the number of lymphocytes plus macrophages was over the median number of these cell types in cChHD, moderate when the number of lymphocytes plus macrophages was between 25th and 50th percentile, and mild when the number of lymphocytes plus macrophages was under percentile 25th.

For quantitative assessment of fibrosis an interventricular septum block that was embedded in paraffin and sectioned at 5 mm was stained with picrosirius red. After obtaining digital images with a digital scanner (UMAX Technologies Inc., USA) at

2 x magnification and a 1200 ppp resolution, the percentage of the surface area occupied by collagen was established by morpho-metric analysis using the digital analysis system Image Pro Plus 4.5 (Media Cybernetics, Silver Spring, USA) [21]. The percentage of fibrosis was semi-quantified as mild (<10%), moderate (10%-20%), or severe (>20%) [22]. Molecular detection of T. cruzi in the same interventricular tissue sections by PCR was previously performed [23]. The presence of T. cruzi was also analyzed by direct observation of intracellular amastigotes.

Immunohistochemistry

Formalin fixed paraffin embedded tissue sections were rehydrated. Heat induced antigen retrieval, incubation time and antibody concentrations were selected following the manufacturer's recommendations. For CD3 (Mouse monoclonal, Santa Cruz Biotechnology, USA), CD8 (Leica Microsystems, Germany), CD68 (Biogenex, USA), CD20 (Biogenex, USA), CD45RA (Biogenex, USA), CD21 (Biogenex, USA), PD-1 (Abcam plc, UK), CD27 (Leica Microsystems, Germany), Ki67 (Rabbit monoclonal from Abcam plc, UK, and mouse monoclonal HLA-G from Leica Microsystems, Germany) and T-bet (BD, Biosciences, USA), heat induced antigen retrieval was done using Antigen Retrieval Citra Plus (Citrate buffer buffer based Ag retrieval solution pH = 6; Biogenex, USA). Antigen retrieval solution EZ-AR2TM (EDTA based retrieval buffer pH = 9; Biogenex, USA) was used to detect CD4 (Leica Microsystems, Germany) and FOXP3 (BD, Biosciences, USA) expression. For CD57 (BD, Biosciences, USA) and CD45RO expression (Biogenex, USA), no antigen retrieval after rehydra-tion was done. Biotinylated anti mouse immunoglobulin G, peroxidase labeled streptavidin, and AEC (3-amino-9-ethyl carbazole) as chromogen were used as secondary detection system (Biogenex, USA). All immunohistochemistry slides were counterstained with hematoxylin. The list of antibodies and clones used, as well the function of each marker are depicted in Table S1 [24-37].

Immunofluorescence

Tissue sections with high inflammation from patients 1 to 4 were selected for double labeling studies by immunofluorescence.

Table 1. Demographic, clinical and pathologic characteristics of study patients.

Case Gender Age Etiology NYHA FC* LVEDDf (mm) LVEF* (%) Heart weight/body weight (g/Kg)) Myocarditis Median number of CD3++CD68+ cells (IQRs)a Fibrosis (%)B Parasite nestsc

1 M 42 cChHD5 IV 63 15 410/75 287 (144-394) 45 Yes

2 F 48 cChHD IV 72 33 420/48.9 116 (79-185) 23 No

3 M 45 cChHD III 75 13 495/61 117 (63-166) 11 No

4 M 46 cChHD III 77 14 460/66.6 72 (42-83) 21 No

5 M 59 cChHD III 65 21 460/54.5 20 (18-54) 12 No

6 F 51 cChHD IV 47 26 555/71 33 (25-47) 7 No

7 F 61 cChHD III 61 14 335/54.7 18 (13-26) 22 No

8 F 59 cChHD IV 76 18 465/51.9 14 (8-26) 20 No

A M 28 GCM11 IV 59 25 395/87 217 (187-266) 31 No

B M 47 GCM IV 61 20 355/105 185 (101-243) 61 No

C M 46 iDCM" IV 78 30 510/79.5 9 (4-12) 11 No

AThe degree of myocarditis was determined according to the number of lymphocytes plus macrophages in cChHD: Severe >48 median CD3++CD68+/HPF, Moderate 20-48 median CD3++CD68+/HPF, Mild <20 median CD3++ CD68+/HPF.

BThe percentage of fibrotic area was determined by morphometric analysis as described in Materials and Methods [21].

cParasite DNA by PCR was detected in heart sections of all cChHD, except for patient 6 [23]. Abbreviations:

* NYHA FC, New York Heart Association, functional class;

^LVEDD, left ventricular end-diastolic diameter;

^LVEF, left ventricular ejection fraction;

§cChHD, chronic Chagas heart disease;

^GCM, Giant Cell Myocarditis;

"iDCM, idiopathic dilated cardiomyopathy;

§IQR, interquartile range; HPF, high power field.

doi:10.1371 /journal.pntd.0002989.t001

Table 2. Phenotypic and functional profile of inflammatory cells in heart tissues from cChHD, GCM and IDCM.

Case CD3 CD8 CD4 CD68 CD20 CD57 PD-1 CD45RO CD27 KÍ67 T-bet FOXP3 HLA-G

1 203/407 122/306 104/397 65/403 12/1 77 3/279 1/291 1 72/282 74/241 40/350 80/296 0/180 0/408

(49.9) (39.9) (26.2) (16.1) (6.8) (1.1) (0.3) (61.0) (30.7) (11.4) (27.0) (0) (0)

2 87/299 85/168 40/198 39/226 1/320 3/285 0/241 75/189 65/199 4/156 5/233 3/340 0/205

(29.1) (50.6) (20.2) (17.3) (0.3) (1.0) (0) (39.7) (32.7) (2.6) (2.1) (0.9) (0)

3 78/1 75 72/209 85/290 37/1 74 3/233 12/224 0/199 82/181 25/169 15/218 56/234 0/130 0/97

(44.6) (34.4) (29.3) (21.3) (1.3) (5.4) (0) (45.3) (14.8) (6.9) (23.9) (0) (0)

4 40/105 99/186 57/200 27/196 1/157 2/82 0/128 60/168 24/94 32/261 3/95 0/185 0/62

(38.1) (53.2) (28.5) (13.8) (0.6) (2.4) (0) (35.7) (8.2) (12.3) (3.2) (0) (0)

5 6/42 26/48 5/43 15/74 0/77 2/44 0/33 15/89 4/65 15/116 22/144 0/70 0/42

(14.3) (54.2) (11.6) (20.3) (0) (4.5) (0) (16.8) (6.1) (12.9) (15.3) (0) (0)

6 14/76 12/37 1/50 22/89 0/56 2/62 0/37 1/64 7/79 0/68 0/50 1/92 0/65

(18.4) (32.4) (2) (24.7) (0) (3.2) (0) (1.5) (8.9) (0) (0) (1.1) (0)

7 5/43 2/18 1/56 14/64 0/48 1/52 0/53 4/52 0/24 0/33 0/50 0/53 0/37

(11.6) (11.1) (1.8) (21.9) (0) (1.9) (0) (7.7) (0) (0) (0) (0) (0)

8 4/42 3/12 0/55 8/53 0/46 1/36 0/31 3/54 0/31 0/44 0/35 0/42 0/37

(9.5) (25) (0) (15.1) (0) (2.8) (0) (5.5) (0) (0) (0) (0) (0)

A 160/358 96/320 102/344 90/322 32/308 0/412 0/174 112/243 112/353 1/447 0/432 0/161 3/455

(44.7) (30) (29.6) (27.9) (10.4) (0) (0) (46.1) (31.7) (0.2) (0) (0) (0.7)

B 68/264 98/383 163/439 112/288 8/263 2/242 1/282 150/386 210/436 4/365 5/250 0/298 52/451

(25.7) (25.6) (37.1) (38.9) (3.0) (0.8) (0.3) (38.9) (48.2) (1.1) (2) (0) (11.5)

C 1/24 0/19 0/16 9/42 0/14 0/36 0/37 0/26 0/31 0/19 0/16 0/15 0/23

(4.2) (0) (0) (21.4) (0) (0) (0) (0) (0) (0) (0) (0) (0)

Data are shown as the median number of positive cells for each marker/median number of total mononuclear cell count in 10 high power field (x400) for each marker for each patient. Percentages are shown in brackets. Chronic Chagas disease patients with advanced heart disease (cChHD) [1-8]; Patients suffering from Giant Cell Myocarditis (GCM) [A and B]; Patient with Idiopathic Dilated Cardiomyopathy (IDCM) [C]. doi:10.1371 /journal.pntd.0002989.t002

Figure 1. Degree of inflammation and cell type composition of inflammatory cell infiltrates in heart samples from patients with chronic Chagas disease. A) The total number of mononuclear inflammatory cells were counted in 10 HPF [Original Magnification (O.M.) 400x] from the interventricular septum of 13 sections assessed for each patient with Chagas disease (n = 8), GCM (n = 2) or DCM (n = 1). Each dot represents the total number of mononuclear inflammatory cells in one out of the 130 HPFs counted for each patient. The horizontal lines indicate median values. Arrows indicate the HPF in which parasites were detected by direct observation. B) Representative photos of immunohistochemistry showing total T cells (CD3+), cytotoxic T cells (CD8+), helper T cells (CD4+), macrophages/dendritic cells (CD68+) and B cells (CD20+). Arrows show positive staining for the corresponding marker. C) Correlation analysis between the percentage of cells expressing each marker and the total number of mononuclear inflammatory cells in chronic Chagas disease patients (n = 8) was performed by Spearman correlation test. doi:10.1371/journal.pntd.0002989.g001

Double labeling assays were carried out by staining with a combination of anti-Ki67 (rabbit monoclonal antibody)/ anti-CD8; anti-KI67/anti-CD20 (mouse mAbs), anti-Ki67 (mouse mAb)/anti-CD3 (rabbit polyclonal) and anti-Ki67/anti-CD21 (rabbit monoclonal) (Table 1). Fluorescein labeled anti-rabbit goat immunoglobulin (Vector, USA) and Alexa fluor 594 labeled anti-mouse immunoglobulin (Invitrogen, USA) were used as secondary detection system. Nuclei staining were done with ready to use mounting medium for fluorescence with DAPI (Vectashield, Vector, USA). Antibody dilutions were used according to the manufacturer's instructions. Observations were made with a 100 W ultraviolet lamp and photographed with an AXIOCAM camera (Carl Zeiss AG, Germany).

Quantification of cells

For quantification of the total number of mononuclear inflammatory cells (i.e. cells with positive and negative staining for each marker assessed), 10 High Power Field (HPF) at 400x were counted for each section (1 section for each one of the 13 markers assessed). The median number of mononu-clear inflammatory cells in 130 HPF was calculated for each patient.

The total number of mononuclear inflammatory cells with positive staining for each marker assessed was counted in 10 HPF, Magnification 400x. The percentage of cells expressing each marker was calculated by the ratio between the median number of positive cells for each marker and the median number of total mononuclear cell count in 10 HPF.

Cell counting was manually implemented using the Cell Counter plug-in of Image J 1.45b software (National Institutes of Health, USA). The position of the cells in each HPF was determined by Cell Counter plug-in and utilized thereafter for spatial histological analysis. This analysis was carried out in each one of the eight samples from Chagas disease patients, as well as in control samples.

Statistical analysis

Continuous variables are reported as means (SDs) or medians (interquartile range -IQR-), while categorical variables are presented as the percentage of subjects out of total subjects evaluated. Continuous variables with non-Gaussian distribution were analyzed by Mann-Whitney U test. Correlation analysis between the percentage of cells positive for each marker and the total number of mononuclear inflammatory cells was done using the Spearman correlation test.

In order to estimate the spatial pattern of infiltrating cells surrounding a T. cruzi -infected cardiomyocyte, a spatial point pattern analysis was applied. A focal analysis of the distribution of CD45RO+ cells around an infected cardiomyocyte was performed by the bivariate Wiegand-Moloney O-ring statistic [38]. This test allows the characterization of a spatial pattern around a point at varying distances to the point, detecting aggregation, repulsion or randomness. The analysis was implemented with software Programita, using a random labeling null model. Confidence

envelopes were calculated using 999 Montecarlo simulations [39]. A p value<0.05 (2-tailed) was considered statistically significant. For statistical analysis SPSS 11.0 statistical software (SPSS Inc, USA) was used.

Results

Clinical and demographical characteristics of cChHD patients

The clinical and demographical characteristics of cChHD patients are depicted in Table 2. Patients 1-5 and 7-8 were in end-stage cChHD, whereas patient 6 displayed cChHD and concomitant valvular heart disease. This patient suffered from rheumatic fever during her childhood, but no evidence of active endocarditis was observed by the time of the study. All patients were in the New York Heart Association classes III and IV by the time of transplantation. Echocardiography studies revealed moderate to severe dilation of the cavities with a mean left ventricular end-diastolic diameter of 67.0610.2 mm. The mean left ventricular ejection fraction, determined by radionuclide ventriculography, was 19.267.1%.

Analysis of cardiac explants specimens

The mean weight of cChHD explanted hearts was 450665 g. Four:8 cChHD interventricular septum samples showed severe diffuse myocarditis, 2:8 moderate myocarditis and 2:8 had mild focal myocarditis. Interstitial fibrosis was moderate to severe in all except for one interventricular septum sample. Amastigote nests were only recorded in 3 sections with severe diffuse myocarditis of patient 1 (Table 1, Figure 1A). However, the presence of T. cruzi by PCR in the interventricular septum of patients 1-5 and 7-8 but not in patient 6 was demonstrated in a previous report [23]. The mean weight of hearts with GCM was 375 g and exhibited severe diffuse myocarditis and severe fibrosis. The IDCM heart weighted 510 g, presenting moderate fibrosis and absence of myocarditis (Table 1).

Characterization of the inflammatory cell infiltrate in the hearts of patients with severe chronic Chagas disease

Inflammatory mononuclear cell infiltrates in heart tissue samples from cChHD were evaluated by immunohistochemistry-As observed in Figure 1A and Table 2, patients 1 to 4 exhibited severe diffuse inflammatory infiltrate, while patients 5 to 8 presented lower number of total infiltrating cells within the interventricular septum. The percentage of T (CD3+, CD4+, CD8+) and B cells (CD20+) was correlated with the total cell count in inflammatory cell infiltrates (Figure 1B and 1C). Conversely, the percentage of macrophages/dendritic cells (CD68+ cells) in regions with the highest levels of infiltration was similar to those observed in areas with low inflammation (Figure 1B and 1C). In 7 out of the 8 cChHD, CD8+ T cells were prevalent among infiltrating mononuclear cells compared to CD4+, CD68+ and CD20+ cells (Table 2).

Figure 2. Expression of markers of T cell differentiation and degree of inflammation in the heart of chronically T. cruzi-infected subjects with severe cardiomyopathy. A-D, left panel: representative photos of CD45RO, CD27, PD-1 and CD57 expression, respectively. A-D, right panel. Each dot represents the percentage of cells expressing each marker (Y axis) vs. the total number of mononuclear inflammatory cells (X axis) in each HPF out 10 HPF (O.M. 400x) evaluated for patient (n = 8). Correlation analysis was done by Spearman correlation test. Arrows in C indicate positive staining for PD-1. E) CD27 expression in the heart from one GCM patient (left panel) and in control lymph node tissues (right panel). F) PD-1 expression in myocardial lymphoid follicles (tertiary lymphoid follicles) in the heart of one chagasic patient (left panel) and in control lymph node tissues (right panel). doi:10.1371/journal.pntd.0002989.g002

T cell differentiation of infiltrating cells in the heart of patients with Chagas disease myocarditis

Since T cells were the prevalent cell population in heart tissues with high degree of inflammation, the expression of markers of antigen-experienced T cells and T cell differentiation (Table S1) was evaluated in heart samples. A high percentage (>30%) of infiltrating cells in heart tissues from cChHD with severe myocarditis express CD45RO, a marker of antigen-experienced T cells (Figure 2A, patients 1-4 in Table 2). Likewise, CD27 expression, a marker of low grade T cell differentiation was higher in cChHD with severe myocarditis than in patients with lower degree of inflammation (Figure 2B, patients 1-4 vs. patients 5-8 in Table 2). In contrast, the expression of the inhibitory receptor PD-1, the immunosenescence marker CD57 and CD45RA (i.e. expressed by naive and terminally differentiated T cells; Table S1), was generally very low in heart tissues from cChHD (Figure 2C, 2D, Figure S1, Table 2). Since CD57 is expressed in terminally differentiated effector T cells and also in NK, the low expression of CD57 confirms the scarcity of NK in heart tissue samples from chronic Chagas disease patients [3]. PD-1 expression was detected in intramyocardial lymphoid follicles (tertiary lymphoid follicles) in the heart of patients with severe Chagas myocarditis (i.e. patients 1-4) [Figure 2C and 2F, left panel]. Of note, tertiary lymphoid follicles were randomly observed in myocardial tissues, as confirmed by the expression of CD21 and PD-1. These findings indicate that antigen-experienced T cells with low grade of differentiation are abundant in Chagas disease myocarditis.

Infected cardiac myocytes are surrounded by cells bearing memory markers

To better characterize the phenotype of the cell infiltrate recruited by infected myocytes, tissue samples bearing amastigotes nests along with high inflammation were selected and analyzed for the expression of CD45RO, CD45RA, CD57, CD68 and CD20 (Figure S1). Most cells surrounding the infected cardiomyocyte were CD45RO+, CD45RA", CD57", CD20"; while a lower number expressed CD68+ (Figure S1A and S1B).

With the aim to evaluate whether the different cell types were recruited with the same efficacy towards the infected cardiomy-ocyte, the spatial pattern of CD45RO+ and CD45RO cells was analyzed by using the O-ring test for 2-dimensional point patterns. A statistically significant aggregation of CD45RO+ cells (blue line in Figure S1D) was observed near the infected cardiomyocyte (i.e. CD45RO+ cells are over the confidence envelope) [Figure S1C and S1D, P<0.001]. The proportion of CD45RO+ cells decreases at longer distances from the infected cell.

Functional profile of T cells in Chagas disease myocarditis: High number of T-bet- and Ki67-expressing lymphocytes and low number of FOXP3+ cells

The functional profile of the cell infiltrate was assessed by the expression of T-bet, a marker of type 1 T cell responses, the regulatory molecules FOXP3 and HLA-G, and Ki67, a marker of proliferative potential (Table S1). T-bet expression was broadly

detected in cell nuclei in areas of high cell infiltrate and was correlated with the degree of cell infiltration (P<0.001, rho = 0.669) (Figure 3A; Table 2). In contrast, HLA-G+ and FOXP3+ cells were found in a very low proportion or were absent in cChHD (Figure 3B, Table 2). Cells with proliferative potential were observed in tissue samples from patients with severe myocarditis (median percentage of proliferating cells = 9%) in areas with high inflammation (Figure 3C, Table 2 and Table 3). In order to identify the proliferating lymphocyte subsets, double immunofluorescence staining was carried out using Ki67, CD3, CD8 and CD21 antibodies (Figure 4). Most cells expressing Ki67+ were CD3+ cells [median percentage (interquartile range) = 74 (66-89)], while a minor proportion of Ki67+ cells expressed CD8 [median percentage (interquartile range) = 21 (15-19)], (Figure 4A, 4B and 4C). In contrast, a very low proportion of Ki67+ cells expressed CD21 (data not shown), suggesting that KI67 /CD21 proliferating cells belong to the T cell compartment.

Comparison of phenotypic and functional profiles between chronic Chagas disease myocarditis and Giant Cell Myocarditis

To assess whether the phenotypic and functional profiles found in Chagas disease myocarditis were associated with parasite persistence, the same set of markers were assessed in heart tissues from patients suffering from idiopathic GCM (patient A and B, Table 2), and compared to the profile in cChHD with severe myocarditis (patients 1-4, Table 2). GCM is characterized by an intense myocarditis with multifocal cardiomyocyte damage. Although T lymphocytes were a major cell type in both severe cChHD and GCM, the latter was enriched in macrophages/ dendritic cells and CD4+ T cells (Table 3), whereas CD8 was prevalent in cChHD with severe myocarditis.

Regarding the differentiation status of the cell infiltrate in these two types of myocarditis, no differences were found in PD-1 and CD45RO expression, whereas CD57 expression was low in both types of myocarditis. cChHD with severe myocarditis presented lower levels of CD27+ cells (i.e. 2/4 patients with severe myocarditis in Table 2) compared to GCM (Table 2 and Table 3). Higher counts of Ki67+ and T-bet+ cells were found in cChHD as compared with GCM, whereas the expression of FOXP3 was very low in both myocarditis. HLA-G expression was only recorded in cardiomyocytes and mononuclear inflammatory cells from GCM (Table 2 and Table 3). Altogether, these findings support the notion that chronic infection with T. cruzi drives T cell differentiation and proliferation in chronic Chagas disease myocarditis.

Discussion

Most studies concerning the characterization of inflammatory cells in heart tissues in chronic Chagas disease have comprised the evaluation of different cell types, and the cytokine profile, while the differentiation status of T cells is less known. Herein, we report that antigen-experienced (CD45RO+) T cells with a low degree of

b « «

^ Wir w, m i " ' -

B ■te ^ w í ^ * " to f '' 9 . ^vbH r3b „ , w / 5. "*«* * • -JÍ f

V / V dir, ' » r» ' - f " - tf "Í. tr & t*v ♦ f "J . • > j i ■>- , « £ * - ^ #

^ ^ Tí 9 * a 4

«j» * * /9 0 S ' / * #

F0XP3 ra * »^r - . wj

« * . >

* >.. .»'IPV,

.jf (•' v ♦ V .

* • % ▼ KÍ67

»r . • >»*

* > ? »S

v * 4 .

s. • . ■1 . ' -

T-bet - v ^

P<0.001 rho=0.669

200 400 600

Number of infiltrating cells/HPF

X 40 O

P=0.034 rho=0.250

••— fcfi

200 400 600 800

Number of infiltrating cells/HPF

P<0.001 rho=0.567

200 400 600

Number of infiltrating cells/HPF

Figure 3. T-bet, FOXP3 and Ki67 expression in cChHD myocarditis. A) Representative photo (O.M. 400x) of T-bet+ cells (arrows) in cChHD, (left panel). Percentage of T-bet+ cells vs. total mononuclear inflammatory cells counted in 10 representative HPFs for each cChHD (n = 8), [right panel]. B) Representative photo (O.M. 400x) of FOXP3 in cChHD, left panel. Arrows indicate FOXP3+ cells. Percentage of FOXP3+ cells vs. total mononuclear inflammatory cells counted in 10 representative HPFs for each cChHD (n = 8), right panel. C) Representative photo (O.M. 400x) of Ki67 in cChHD, left panel. Arrows indicate Ki67+ lymphocytes in cChHD. Percentage of Ki67+ cells vs. total mononuclear inflammatory cells counted in 10 representative HPFs for each cChHD (n = 8), right panel. Correlation analysis for all markers was carried out by Spearman correlation test. doi:10.1371/journal.pntd.0002989.g003

Table 3. Comparative analysis between inflammatory cell infiltrates of cChHD and GCM.

Markers cChHDsM (n = 4) GCM(n = 2) P value cChHDsM vs. GCM

Cell type

CD3 87/206 (42.2) 137/330 (41,5) 0.374

CD8 92/205 (44.9) 97/269 (36.1) 0.483

CD4 76/272 (27.9) 132/392 (33.7) 0.009

CD68 36/224 (16.1) 101/312 (32.4) <0.001

CD20 3/211 (1.4) 7/275 (2.5) 0.147

Differentiation

CD57 3/222 (1.3) 2/313 (0.6) 0.001

PD-1 0/231 (0) 1/229 (0.4) 0.146

CD45RO 82/203 (40.4) 120/355 (33.8) 0.541

CD27 45/187 (24.1) 154/425 (36.2) <0.001

Functional

Ki67 16/222 (7.2) 1/408 (0.2) <0.001

T-bet 30/206 (14.6) 1/426 (0.2) <0.001

FOXP3 0/1 82 (0) 0/261 (0) 0.092

HLA-G 0/1 37 (0) 24/455 (5.3) <0.001

Data are shown as the median number of positive cells for each marker/median number of total mononuclear cell count in 10 HPF (400x) for each marker. Percentages are shown in brackets.

Significant differences between groups were assessed by Mann- Whitney U test. cChHDsM, chronic Chagas heart disease with severe myocarditis (patients 1-4 of

Table 3); GCM, Giant Cell Myocarditis; HPF, high power field.

doi:10.1371/journal.pntd.0002989.t003

differentiation (CD27+/CD572), a Th1 profile (Tbet+) and proliferative capacity (Ki67+) are recruited into the heart of patients with chronic advanced Chagas disease myocarditis. As previously described, the inflammatory cell infiltrate in most patients was dominated by CD8+ T cells [2,3,6]. Although less prominent than CD8+ T cells, appreciable counts of CD4+ T cells were also found in intracardiac infiltrates.

The T cell phenotype in heart tissues concurs with that observed in circulating CD8+ and CD4+ T cells responsive to T. cruzi antigens in patients with chronic Chagas disease, regardless the clinical status [15,16], but it is at odds with the phenotype of total peripheral CD4+ and CD8+ T cells [15-17], [Table S2, 8,1013,15-17,40-43]. Whereas most circulating IFN-y-producing CD8+ and CD4+ T cells responsive to T. cruzi display a low degree of differentiation (CD27+/CD28+/CD572/LIR-12) [8,16], high differentiated (cD27"/CD28"/CD57+/LIR-1+) CD8+ and CD4+ T cells are increased in the total peripheral T cell compartment of patients with severe cardiomyopathy [8,1517]. Subjects with chronic T. cruzi infection with severe cardiomyopathy also displayed lower frequencies of T. cruzi-responsive IFN-y-producing T cells and lower levels of IFN-y production compared to subjects with no signs of heart disease [8,13,16], leading us to propose that long-term parasite persistent might dampen parasite specific T cell responses [44]. Thus, recently developed effector T cells that bear a low degree of differentiation appear to be an important source of T. cruzi-specific T cells in the periphery of chronic Chagas disease patients. In contrast, the bulk of the total CD8+ and CD4+ T cells might reflect the effect of persistent exposure to the parasite.

The low degree of differentiation of T cells found in the heart of chronically T. cruzi-infected subjects was somehow unexpected, since T cells at target tissues are more likely to be stimulated by antigen and induced to further differentiation. However, CD272

T cells also comprised a substantial fraction of the inflammatory infiltrate, indicating a heterogeneous composition of T cells with different degree of differentiation in heart tissues. The presence of CD27+/CD57 cells might account for proliferating CD3+ T cells, as proliferation is a hallmark of T cells with low grade of differentiation (CD27+ cells) and low rounds of antigen stimulation (CD57" T cells) [45].

We also demonstrate that antigen-experienced (CD45RO+) T cells were more efficiently recruited than other mononuclear cells towards the infected cardiomyocyte, supporting that T. cruzi is a driving force for T cell recruitment. Activated VLA-4+/LFA-1+/granzyme A+ T lymphocytes have been observed in cardiac infiltrates of patients with chagasic heart failure [7], while the local production of IL-7 and IL-15 was claimed to be associated with the maintenance and predominance of CD8+ T cells in heart tissues [46]. However, the recruitment of early effector T cells might be also involved in the maintenance of inflammatory cells at sites of chronic localized infection.

Tertiary lymphoid organs associated to severe myocarditis might have been developed in the context of chronic inflammatory conditions [47-49]. Since the formation of TLO involves the recruitment of lymphocytes not normally associated with inflammatory infiltrates, notably naive T cells and central memory T cells, these structures might be a source of recently recruited effector T cells from the naïve or central memory pool. To the best of our knowledge, there are not reports describing these structures in chronic chagasic cardiomyopathy. TNF-a has been pointed out as a key molecule in the generation of ectopic TLO which might facilitate the perpetuation of inflammation in the heart. Cells expressing TNF-a and IFN-y have been identified in heart tissues from patients with chronic chagasic cardiomyopathy [9,50]. Our findings showing T-bet expression in areas of high inflammation and proliferation further confirmed a type 1 T cell response in

123456789 10 123456789 10

HPF Number HPF Number

Figure 4. Proliferation of T and B cells in Chagas disease myocarditis. A) Representative photos (O.M. 400 x) of double immunofluorescence staining of Ki67 with CD3 (upper row); CD8 (middle row); or CD21 (lower row) cell. All images correspond to patient 1. Nuclei were stained with DAPI (blue). Single CD3 (green), single Ki67 (red) and double CD3 and Ki67 staining (merge) are shown in the upper row (inset, arrows). Single CD8 (red), single Ki67 (green) and double CD8 and Ki67 staining are shown in the middle row (inset, arrow). Single CD21 (green) and single Ki67 (red) expression is shown in the lower row (inset, arrow). Proportion of total Ki67+ cells showing CD3 (B) or CD8 (C) expression (filled columns), in 10 HPFs (numbered from 1 to 10) of patient 1. doi:10.1371/journal.pntd.0002989.g004

patients with severe heart disease. The low levels of PD-1 counts concur with the presence of T-bet+ cells, since T bet inhibits PD-1 expression [51].

Recent reports have demonstrated that regulatory T cells (FOXP3+) are absent in Chagas disease myocarditis, indicating that deficiency in IL-10 producing T regs may led to a regulatory imbalance, perhaps rendering heart tissues increasingly susceptible to type 1-dependent pathology [9,52]. In line with these findings, scarce FOXP3 cells were found in heart tissues from patients with severe heart disease. Nevertheless, we have shown that CD3+ T lymphocytes infiltrating heart tissues express CTLA-4, another negative regulator of effector T cell responses [8]. In addition to be involved in the regulatory T cell function of T regs, CTLA-4 is also upregulated on activated T cells [53]. This might explain the concomitant presence of proliferating and CTLA-4+ T cells. However, it is likely that proliferating T lymphocytes do not CTLA-4 or likely express dysfunctional CTLA-4.

The predominant role for T. cruzi in the differentiation and functional T cell profile observed in the heart of subjects with chronic T. cruzi infection becomes apparent at comparing the inflammatory cell infiltrates between chagasic and GCM myocarditis which is a rare and frequently fatal type of myocarditis with features of autoimmune disease [54,55]. Higher levels of infiltrating cells with low degree of differentiation (i.e. CD27 expressing cells), lower T-bet expression and lower proliferative capacity were observed in GCM compared with severe Chagas disease myocarditis. Of note, HLA-G, a non-classical class I major histocompatibility complex molecule playing a tolerogenic role in innate and adaptive responses [56] was not expressed in inflammatory cell infiltrates of chronic Chagas disease patients, while it was expressed by cardiomyo-cytes in GCM. These findings support that immunoregulation is different between an infectious and a non-infectious myocarditis. Coxsackievirus infection is another common cause of infectious myocarditis, in which a pathogen driven inflammatory process was supported by the reduction in inflammation and resolution of the infection after treatment with IFN-beta [57,58].

The development of tissue damage might depend on parasite burden; the effectiveness of the host immune response in controlling parasite replication, and the effectiveness of the host immune response in limiting peripheral damage. The effectiveness of the host immune response in controlling parasite replication without the induction of tissue damage will largely rely on the quality of T cell responses [59]. We have recently reported that children in early stages of T. cruzi infection maintain polyfunctional and stronger T cell responses to T. cruzi in their circulation, contrasting with the prevalent monofunctional T cell profile in long-term T. cruzi-infected adults [60]. Although, we cannot rule out that recruitment of IFN-y-producing T cells into the heart might account for the decrease of IFN-y-producing T cells from the periphery, it is also possible that clonal exhaustion occurs overtime. Consequently, in the phase of an ineffective T cell response, the development of tissue damage might occur in order to get parasites under control. Similarly, immunoregulatory pathways might be also dampened overtime.

In summary the quality of T cell responses and immunoreg-ulatory mechanisms might determine the pattern of the

References

1. Rassi A Jr., Rassi A, Marin-Neto jA (2010) Chagas disease. Lancet 375: 13881402.

cellular response and the severity of disease in chronic T. cruzi infection.

Limitation of the study

The main limitation of the study is the sample size. However, the detailed analysis conducted and the difficulty to get tissue samples can mitigate, at least partially, such limitation.

Supporting Information

Figure S1 Spatial analysis of infiltrating cells surrounding T. cruzi-infected cardiomyocyte in heart tissues from patients with chronic Chagas disease. A) Immunohistochemistry staining of the infected cardiomyocyte showing CD45RO, CD45RA, CD57 and CD68 expression [O.M. 400 x]. Open arrows show amastigote nests while black arrows indicate positive staining for the corresponding marker. B) Immunohistochemistry assay [O.M. 100 x] showing antigen-experienced CD45RO+T cells surrounding the infected cardiomyocyte. C) Spatial analysis of CD45R0+ T cells surrounding an infected human cardiomyocyte. Cell positioning dot plot showing 933 CD45RO+ (red dots) and 428 CD45RO (blue dots) T cells. D) Statistics of the spatial distribution of CD45RO+ cells at different distances (r) from the infected cardiomyocyte was performed using the O-ring test [2425]. The statistic values are depicted as O12 (r) (blue line). The confidence envelopes (P<0.001, grey lines, E12+ and E12 ) were determined by 999 Monte Carlo simulations. The upper limit of the confidence envelope (upper grey line, E12+) delimits statistically significantly aggregation of CD45RO cells; while the lower limit (E12 " ) delimits statistically significant repulsion of these cells. Values between E12+ and E12 do not differ significantly from randomness. (TIF)

Table S1 Note: * Unless otherwise stated, antibodies were unconjugated mouse monoclonal antibodies. { CD8a is also expressed in some dendritic cell subsets, and can be induced in NKs and HDE CD4 T cells. { CD4 is expressed by monocytes and can be induced in macrophages and dendritic cells. DE, Terminally Differentiated Effector T cells; TCM, Central Memory T cells; TEM, Effector Memory T cells. (DOCX)

Table S2 A T. cruzi-infected vs. uninfected controls. B PBMC were stimulated with T. cruzi antigens. E, effector; CM, central memory; EM, effector memory; TET, terminally differentiated effector; cChHD, advanced chronic Chagas heart disease. (DOCX)

Acknowledgments

We thank Marcela Alvarez (Hospital Universitario Fundacion Favaloro, Bs As) for histological preparation of samples.

Author Contributions

Conceived and designed the experiments: SAL CV. Performed the experiments: RJA PCM. Analyzed the data: SAL CV RJA RV FG LEF RRF. Contributed reagents/materials/analysis tools: SAL RL FG LEF RRF. Wrote the paper: SAL CV RJA RL.

2. Higuchi ML, Martins Reis M, Barbosa A, Bellotti G, Pereira-Barreto AC, et al. (1993) Correlation between Trypanosoma cruzi parasitism and myocardial

inflammatory infiltrate in human chronic chagasic myocarditis: Light microscopy and immunohistochemical findings. Cardiovasc Pathol 2: 101— 106.

3. Higuchi ML, Gutierrez PS, Aiello VD, Palomino S, Bocchi E, et al. (1993) Immunohistochemical characterization of infiltrating cells in human chronic chagasic myocarditis: comparison with myocardial rejection process. Virchows Arch A Pathol Anat Histopathol 423: 157-160.

4. Tarleton RL. Parasite persistence in the aetiology of Chagas disease (2001) IntJ Parasitol 31: 550-554.

5. Schijman AG, Vigliano CA, Viotti RJ, Burgos JM, Brandariz S, et al. (2004) Trypanosoma cruzi DNA in cardiac lesions of Argentinean patients with endstage chronic chagas heart disease. AmJ Trop Med Hyg 70: 210-220.

6. Reis DD, Jones EM, Tostes S Jr., Lopes ER, Gazzinelli G, et al. (1993) Characterization of inflammatory infiltrates in chronic chagasic myocardial lesions: presence of tumor necrosis factor-alpha+ cells and dominance of granzyme A+, CD8+ lymphocytes. AmJ Trop Med Hyg 48: 637-644.

7. D'Avila Reis D, Jones EM, Reis Lopez STE, Chapadeiro E, Gazzinelli G, et al. (1993) Expression of major histocompatibility complex antigens and adhesion molecules in hearts of patients with chronic Chagas disease. AmJ Trop Med Hyg 49: 192-200.

8. Arguello RJ, Albareda MC, Alvarez MG, Bertocchi G, Armenti AH, et al. (2012) Inhibitory receptors are expressed in Trypanosoma cruzi-specific T cells and in the hearts of subjects with chronic Chagas disease. Plos One 7: e35966.

9. Rocha Rodrigues DB, dos Reis MA, Romano A, Pereira SA, Teixeira V de P, et al.(2012) In situ expression of regulatory cytokines by heart inflammatory cells in Chagas' disease patients with heart failure. Clin Dev Immunol 2012: 361730.

10. Abel LC, Rizzo LV, Ianni B, Albuquerque F, Becal F, et al. (2001) Chronic Chagas disease cardiomyopathy patients display an increased IFN-gamma response to Trypanosoma cruzi infection. J Autoimmun 17: 99-107.

11. Gomes JAS, Bahia-Oliveira LMG, Rocha MOC, Martins-Filho OA, Gazzinelli G, et al. (2003) Evidence that development of severe cardiomyopathy in human Chagas' disease is due to a Th1-specific immune response. Infect Immun 71:1185-93.

12. Lorena VM, Lorena IM, Braz SC, Melo AS, Melo MF, et al. (2010) Cytokine levels in serious cardiopathy of Chagas disease after in vitro stimulation with recombinant antigens from Trypanosoma cruzi. Scand J Immunol 72: 529-39.

13. Laucella SA, Postan M, Martin D, Hubby Fralish B, Albareda MC, et al. (2004) Frequency of interferon-gamma-producing T cells specific for Trypanosoma cruzi inversely correlates with disease severity in chronic human Chagas disease. J Infect Dis 189: 909-918.

14. de Melo AS, de Lorena VM, de Moura Braz SC, Docena C, de Miranda Gomes Y. (2012) IL-10 and IFN-y gene expression in chronic Chagas disease patients after in vitro stimulation with recombinant antigens of Trypanosoma cruzi. Cytokine 58:207-12.

15. Albareda MC, Laucella SA, Alvarez MG, Armenti AH, Bertochi G, et al. (2006) Trypanosoma cruzi modulates the profile of memory CD8+ T cells in chronic Chagas' disease patients. Int Immunol 18: 465-471.

16. Albareda MC, Olivera GC, Laucella SA, Alvarez MG, Fernandez ER, et al. (2009) Chronic human infection with Trypanosoma cruzi drives CD4+ T cells to immune senescence. J Immunol 183: 4103-4108.

17. Dutra WO, Martins-Filho OA, Cancado JR, Pinto-Dias JC, Brener Z, et al. (1996) Chagasic patients lack CD28 expression on many of their circulating T lymphocytes. Scand J Immunol 43: 88-93.

18. Aretz HT, Billingham ME, Edwards WD, Factor SM, Fallon JT, et al. (1987) Myocarditis. A histopathologic definition and classification. Am J Cardiovasc Pathol 1: 3-14.

19. Edwards WD (1984) Myocarditis and endomyocardial biopsy. Cardiol Clin 2:647-656.

20. Caforio AL, Pankuweit S, Arbustini E, Basso C, Gimeno-Blanes J, et al. (2013) Current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases. Eur Heart J 34:263648, 2648a-2648d.

21. Fernandez A, Vigliano CA, Casabe JH, Diez M, Favaloro LE, et al. (2011) Comparison of prevalence, clinical course, and pathological findings of left ventricular systolic impairment versus normal systolic function in patients with hypertrophic cardiomyopathy. Am J Cardiol 108:548-55.

22. Kunkel B, Kober G, Lapp H, Leuschner U, Kaltenbach M (1977) Clinical light and electron microscopic findings in patients with late and early congestive cardiomyopathies. Z Kardiol 66: 198-202.

23. Burgos JM, Diez M, Vigliano C, Bisio M, Risso M, et al. (2010) Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation. Clin Infect Dis 51: 485-495.

24. Tsoukas CD, Landgraf B, Bentin J, Valentine M, Lotz M, et al. (1985) Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes. J Immunol 135: 1719-1723.

25. Marrack P, Kappler J (1987) The T cell receptor. Science 238: 1073-1079.

26. Davey FR, Cordell JL, Erber WN, Pulford KA, Gatter KC, et al. (1988) Monoclonal antibody (Y1/82A) with specificity towards peripheral blood monocytes and tissue macrophages. J Clin Pathol 41: 753-758.

27. Cragg MS, Walshe CA, Ivanov AO, Glennie MJ (2005) The biology of CD20 and its potential as a target for mAb therapy. Curr Dir Autoimmun 8: 140-174.

28. Weis JJ, Tedder TF, Fearon DT (1984) Identification of a 145,000 Mr membrane protein as the C3d receptor (CR2) of human B lymphocytes. Proc Natl Acad Sci (USA) 81: 881-885.

29. Mitsumoto Y, Oka S, Sakuma H, Inazawa J, Kawasaki T (2000) Cloning and chromosomal mapping of human glucuronyltransferase involved in biosynthesis of the HNK-1 carbohydrate epitope. Genomics 65: 166-173.

30. Yu H, Shahsafaei A, Dorfman DM (2009) Germinal-center T-helper-cell markers PD-1 and CXCL13 are both expressed by neoplastic cells in angioimmunoblastic T-cell lymphoma. Am J Clin Pathol 131: 33-41.

31. Good-Jacobson KL, Szumilas CG, Chen L, Sharpe AH, Tomayko MM, et al. (2010) PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells. Nat Immunol 11: 535-542.

32. Deans JP, Serra HM, Shaw J, Shen YJ, Torres RM, et al. (1992) Transient accumulation and subsequent rapid loss of messenger RNA encoding high molecular mass CD45 isoforms after T cell activation. J Immunol 148: 18981905.

33. van Lier RAW, Borst J, Vroom TM, Klein H, van Mourik P, et al. (1987) Tissue distribution and biochemical and functional properties ofTp55 (CD27), a novel T cell differentiation antigen. J Immunol 139: 1589-1596.

34. Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, et al. (2000) A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655-669.

35. Bennett CL, Christie J, Ramsdell F, et al. (2001) The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27: 20-21.

36. Carosella ED, Moreau P, Le Maoult J, et al. (2003) HLA-G molecules: from maternal-fetal tolerance to tissue acceptance. Adv Immunol 81: 199-252.

37. Gerdes J, Schwab U, Lemke H, et al. (1983) Production ofa mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31: 13-20.

38. Wiegand T MK (2004) Rings, circles, and null-models for point pattern analysis in ecology. Oikos 104: 209-229.

39. Stockholm D, Benchaouir R, Picot J, Rameau P, Neildez TM, et al. (2007) The origin of phenotypic heterogeneity in a clonal cell population in vitro. Plos One 2: e394.

40. Fiuza JA, Fujiwara RT, Gomes JA, Rocha MO, Chaves AT, et al.(2009) Profile of central and effector memory T cells in the progression of chronic human Chagas disease. PLoS Negl Trop Dis 3: e512.

41. Voltarelli JC, Donadi EA, Carvalho IF, Falcao RP (1990) Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease. Rev Inst Med Trop Sao Paulo 32: 240-8.

42. Menezes CA, Rocha MO, Souza PE, Chaves AC, Gollob KJ, et al. (2004) Phenotypic and functional characteristics of CD28+ and CD28- cells from chagasic patients: distinct repertoire and cytokine expression. Clin Exp Immunol 137:129-38.

43. Alvarez MG, Postan M, Weatherly DB, Albareda MC, Sidney J, et al. (2008) HLA class I-T cell epitopes from trans-sialidase proteins reveal functionally distinct subsets ofCD8 T cells in chronic Chagas disease. PLoS Negl Trop Dis 2 (9):e288.

44. Gigley JP, Bhadra R, Moretto MM, Khan IA (2012) T cell exhaustion in protozoan disease. Trends Parasitol 28: 377-384.

45. Appay V, Rowland-Jones SL (2004) Lessons from the study of T-cell differentiation in persistent human virus infection. Semin Immunol 16: 205-212.

46. Fonseca SG, Reis MM, Coelho V, Nogueira LG, Monteiro SM, et al. (2007) Locally produced survival cytokines IL-15 and IL-7 may be associated to the predominance of CD8+ T cells at heart lesions of human chronic Chagas disease cardiomyopathy. Scan J Immunol 66:362.371

47. PoberJS, Sessa WC. Evolving functions of endothelial cells in inflammation. Nat Rev Immunol 2007; 7: 803-815.

48. Bende RJ, van Maldegem F, van Noesel CJ (2009) Chronic inflammatory disease, lymphoid tissue neogenesis and extranodal marginal zone B-cell lymphomas. Haematologica 94:1109-23.

49. Neyt K, Perros F, GeurtsvanKessel CH, Hammad H, Lambrecht BN, et al. (2012) Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol 33: 297-305.

50. Pissetti CW, Correia D, de Oliveira RF, Llaguno MM, Balarin MA, et al. (2011) Genetic and functional role of TNF-alpha in the development Trypanosoma cruzi infection. PLoS Negl Trop Dis 5 (3):e976.

51. Kao C, Oestreich KJ, Paley MA, Crawford A, Angelosanto JM, et al. (2011) Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and sustains virus-specific CD8+ T cell responses during chronic infection. Nat Immunol 12: 663-671.

52. Guedes PM, Gutierrez FR, Silva GK, Dellalibera-Joviliano R, Rodrigues GJ, et al. (2012) Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent ofcardiomyopathy in human Chagas' disease. PLoS Negl Trop Dis 6: e1630.

53. Alegre ML, Frauwirth KA, Thompson CB (2001) T-cell regulation by CD28 and CTLA-4. Nat Rev Immunol 1: 220-228.

54. Cooper LT Jr, Berry GJ, Shabetai R (1997). Idiopathic giant-cell myocarditis-natural history and treatment. Multicenter Giant Cell Myocarditis Study Group Investigators, N Engl J Med 336: 1860-1866.

55. Rosenstein ED, Zucker MJ, Kramer N (2000) Giant cell myocarditis: most fatal ofautoimmune diseases. Semin Arthritis Rheum 30: 1-16.

56. Carosella ED, Gregori S, Rouas-Freiss N, LeMaoultJ, Menier C, et al.(2011) The role of HLA-G in immunity and hematopoiesis. Cell Mol Life Sci 68: 353— 368.

57. Marchant DJ, BoydJH, Lin DC, Granville DJ, Garmaroudi FS, et al. (2012) Inflammation in Myocardial Diseases. Circ Res 110: 126—144.

58. Chapman NM, Kim KS (2008) Persistent coxsackievirus infection: enterovirus persistence in chronic myocarditis and dilated cardiomyopathy. Curr Top Microbiol Immunol 323: 275-292.

59. Seder RA, Darrah PA, Roederer M (2008) T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol 8:247—58. Erratum in: Nat Rev Immunol 8:486.

60. Albareda MC, De Rissio AM, Tomas G, Serjan A, Alvarez MG, et al. (2013) Polyfunctional T cell responses in children in early stages of chronic Trypanosoma cruzi infection contrast with monofunctional responses of long-term infected adults. PLoS Negl Trop Dis 7(12):e2575.

Copyright of PLoS Neglected Tropical Diseases is the property of Public Library of Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.