Trypanosoma cruzi-induced activation of functionally distinct αβ and γδ CD4- CD8- T cells in individuals with polar forms of Chagas' disease.

Abstract

CD4(-) CD8(-) (double-negative [DN]) T cells have recently been shown to display important immunological functions in human diseases. They express γδ or αβ T-cell receptors that recognize lipid/glycolipid antigens presented via the nonclassical major histocompatibility complex molecules of the CD1 family. We recently demonstrated that while αβ DN T cells serve primarily to express inflammatory cytokines, γδ DN T cells express mainly interleukin-10 (IL-10) in patients with cutaneous leishmaniasis. We also demonstrated a correlation between DN T cells and the expression of gamma interferon in the acute phase of Trypanosoma cruzi experimental infection. In this work, we sought to investigate whether αβ or γδ DN T cells display distinct immunoregulatory potentials in patients with polar forms of human Chagas' disease. Our data showed that in vitro infection with T. cruzi leads to expansion of DN T cells in patients with the indeterminate and severe cardiac clinical forms of the disease. However, while αβ DN T cells primarily produce inflammatory cytokines in both forms of the disease, γδ DN T cells display a marked, significant increase in antigen-specific IL-10 expression in indeterminate patients relative to cardiac patients. Finally, higher frequencies of the IL-10-producing γδ DN T cells were correlated with improved clinical measures of cardiac function in the patients, suggesting a protective role for these cells in Chagas' disease. Taken together, these data show distinct functional characteristics for αβ and γδ DN T cells associated with distinct morbidity rates and clinical forms in human Chagas' disease.

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INFECTION AND IMMUNITY, Oct. 2010, p. 4421–4430
0019-9567/10/$12.00doi:10.1128/IAI.00179-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 10
Trypanosoma cruzi-Induced Activation of Functionally Distinct
?? and ?? CD4?CD8?T Cells in Individuals with Polar
Forms of Chagas’ Disease?
Fernanda Nobre Amaral Villani,1Manoel Ota ´vio da Costa Rocha,2Maria do Carmo Pereira Nunes,2
Lis Ribeiro do Valle Antonelli,3Luisa Moura ˜o Dias Magalha ˜es,1Janete Soares Coelho dos Santos,1
Kenneth J. Gollob,4,5,6and Walderez O. Dutra1,6*
Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais,1and Infectious Diseases and
Tropical Medicine Graduate Course, School of Medicine, Federal University of Minas Gerais,2Belo Horizonte, Minas Gerais,
Brazil; Instituto Oswald Cruz, FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil3; SRI International, Biosciences Division,
Center for Infectious Disease Research, Menlo Park, California4; Graduate Program in Biosciences and Medicine,
Santa Casa Hospital, Belo Horizonte, Minas Gerais, Brazil5; and INCT-DT, Brazil6
Received 22 February 2010/Returned for modification 29 March 2010/Accepted 27 July 2010
CD4?CD8?(double-negative [DN]) T cells have recently been shown to display important immunological
functions in human diseases. They express ?? or ?? T-cell receptors that recognize lipid/glycolipid antigens
presented via the nonclassical major histocompatibility complex molecules of the CD1 family. We recently
demonstrated that while ?? DN T cells serve primarily to express inflammatory cytokines, ?? DN T cells
express mainly interleukin-10 (IL-10) in patients with cutaneous leishmaniasis. We also demonstrated a
correlation between DN T cells and the expression of gamma interferon in the acute phase of Trypanosoma cruzi
experimental infection. In this work, we sought to investigate whether ?? or ?? DN T cells display distinct
immunoregulatory potentials in patients with polar forms of human Chagas’ disease. Our data showed that in
vitro infection with T. cruzi leads to expansion of DN T cells in patients with the indeterminate and severe
cardiac clinical forms of the disease. However, while ?? DN T cells primarily produce inflammatory cytokines
in both forms of the disease, ?? DN T cells display a marked, significant increase in antigen-specific IL-10
expression in indeterminate patients relative to cardiac patients. Finally, higher frequencies of the IL-10-
producing ?? DN T cells were correlated with improved clinical measures of cardiac function in the patients,
suggesting a protective role for these cells in Chagas’ disease. Taken together, these data show distinct
functional characteristics for ?? and ?? DN T cells associated with distinct morbidity rates and clinical forms
in human Chagas’ disease.
T-cell activation is a key event in the establishment of im-
mune responses directed toward intracellular pathogens. De-
pending on the functional capacity of the activated T cells, the
fate of the infection may take different paths either toward a
protective or a pathogenic outcome. While it is important that
a strong, activated immune response is elicited early on in the
infection in order to eliminate (or control) the pathogen, the
further control of this activation is necessary to reestablish
homeostasis, avoiding tissue damage (17, 25).
One hallmark of most parasitic infections is that the great
majority of individuals are able to trigger innate immunity and
elicit an activated T-cell response during the acute infection,
leading to the control of the parasite and establishment of a
chronic infection. Interestingly, while many individuals develop
severe forms of parasitic diseases once infection progresses to
the chronic phase, most patients develop relatively mild forms,
allowing for a host-parasite coexistence. One such example is
observed upon human infection with the protozoan parasite
Trypanosoma cruzi, which leads to Chagas’ disease. As a result
of thousands of years of coevolution between human host and
the parasite (6), most infected individuals develop an asymp-
tomatic, or “indeterminate” (I), form of Chagas’ disease. This
form is characterized by a lack of clinical signs and symptoms
and has been associated predominantly with a modulatory cel-
lular immune response based on cytokine profiles and down-
regulatory molecule expression (5, 20, 48, 49, 51). Chronic
patients may also develop symptomatic clinical forms, mainly
with digestive or cardiac alterations. Differential geographical
prevalence of Chagas’ disease clinical forms has been reported.
In Brazil, 15 to 30% of Chagas’ patients display the cardiac
form, which is present in 20 states, while the digestive cases,
observed in about 10% of infected individuals, have been re-
ported in four states in the central region of the country (53).
The digestive form is frequently found in Chile but is practi-
cally absent in Central America (42). These geographical dif-
ferences might be related, in part, to host genetics and immune
responses of local human populations, but it is believed that
they are also related to the genetic diversity of T. cruzi strains
(11). Different strains of parasite display tropism for different
tissues, and, thus, an important factor determining the clinical
course of disease might be the specific pool of infecting clones
and their specific tropisms (29). However, a possible role for
environmental, nutritional, and immunological aspects of the
* Corresponding author. Mailing address: Laborato ´rio de Biologia
das Interac ¸o ˜es Celulares, Bloco N3, Sala 302, Departamento de Mor-
fologia do Instituto de Cie ˆncias Biolo ´gicas da Universidade Federal de
Minas Gerais, Avenida Anto ˆnio Carlos, 6627, Pampulha, CEP 31270-
901, Belo Horizonte-MG, Brazil. Phone: 55 31 34092809. Fax: 55 31
34092655. E-mail: waldutra@gmail.com.
?Published ahead of print on 9 August 2010.
4421

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host cannot be discounted. While digestive and cardiac forms
present significant morbidity, the cardiac form is the one as-
sociated with highest mortality. It is caused by neuronal and
cardiomyocyte damage, ultimately resulting in ventricular dilation
and subsequent functional heart failure, which can lead to death
(44). Cardiac patients display a T-cell-mediated inflammatory
response in situ (13, 24, 41), which is responsible for the pathol-
ogy; this inflammatory profile is also observed in circulating acti-
vated T cells found at high frequencies in these patients (2, 16, 19,
32). Although it is clear that a plethora of parasite and host
factors influences the clinical outcome of Chagas’ disease, recent
studies have suggested that activation of functionally distinct T-
cell populations in T. cruzi-infected individuals may be respon-
sible for the establishment of different clinical forms (17,
20). Thus, identifying these populations and the factors re-
sponsible for their activation will be critical for driving im-
mune-based interventions to prevent pathology.
While the great majority of T cells express either the CD4 or
the CD8 molecules, which are important for stabilizing the
peptide-major histocompatibility complex (MHC) complex
and which favor T-cell activation, a minority population of T
cells that do not express CD4 or CD8 molecules has been
identified in humans (8, 10, 27, 37). These double-negative
(DN) T cells have been shown to be important sources of
immunoregulatory cytokines in human leishmaniasis (4), to
display modulatory functions (38), but also, under different
circumstances, to display cytolytic activity (10, 36). A subpopu-
lation of DN T cells is activated through the engagement of ??
or ?? T-cell receptors (TCRs) in the recognition of nonclassi-
cal MHC molecules of the CD1 family, presenting lipid or
glycolipid antigens (36). This particular lipid/glycolipid anti-
genic recognition, as well as the immunoregulatory potential
and susceptibility to chronic stimulation of these cells, high-
lights the important role these cells play in parasitic infections.
In our work with Bottrel et al., we determined that DN
lymphocytes were the second most prevalent cell type produc-
ing gamma interferon (IFN-?) in human cutaneous leishman-
iasis and that this IFN-? production was seen after short-term
cultures with medium alone, as well as after stimulation with
soluble Leishmania antigen (SLA) (9). The novel work of An-
tonelli et al. went on to demonstrate that DN T cells composed
of two different cell populations are present in the blood of
individuals infected with Leishmania braziliensis and that DN T
cells expressing the ?? TCR displayed a profile consistent with
activation of leishmanicidal and inflammatory activities (higher
IFN-? and tumor necrosis factor alpha [TNF-?]) while the DN
subpopulation expressing ?? TCR had a modulatory potential
via higher production of interleukin-10 (IL-10) (4). Interest-
ingly, IFN-? production has been associated with pathogenic
responses in human leishmaniasis in more than one clinical
form (3, 7, 22). We recently demonstrated that rats infected
with the CL-Brenner clone of T. cruzi displayed a marked
increase in the frequency of circulating DN T cells during the
acute phase of infection (33). Taken together, these data led to
the question of the role that DN T-cell subpopulations play in
the clinical dichotomy of chronic human Chagas’ disease.
To answer these questions, we investigated the immuno-
regulatory potential of DN T cells in patients with the two
polar forms of Chagas’ disease: indeterminate (I) and dilated
cardiac (DC). Our data demonstrated that although no quan-
titative differences were seen with regard to the nonstimulated
frequency of DN ?? and ?? T-cell subpopulations between
patients and nonchagasic individuals, in vitro infection with
trypomastigote forms of T. cruzi induced a marked increase in
the frequency of these cells from chagasic patients. Moreover,
the expanded ?? DN T cells displayed a greater inflammatory
potential from cardiac patients than from indeterminate pa-
tients. This was accompanied by a greater down-modulatory
ratio of IL-10 to inflammatory cytokine frequencies by ?? DN
T cells from individuals with indeterminate disease, suggesting
distinct roles for these cells in modulating the response in
chronic Chagas’ disease. Finally, we observed a correlation
between higher frequencies of IL-10-producing ?? DN T cells
and improved clinical measures of cardiac function, suggesting
a protective role for these cells in human Chagas’ disease.
These data indicate that functionally distinct DN T cells are
present in Chagas’ disease patients and that they are associated
with the resulting morbidity of the disease.
MATERIALS AND METHODS
Patients. This study employed a cross-sectional design involving patients from
areas of endemicity within Minas Gerais, Brazil, under the medical care of
Manoel O. D. C. Rocha. A total of 12 patients with positive specific serology for
T. cruzi, within the chronic phase of the disease, and with well-defined clinical
forms were enrolled in this study. Detailed evaluations, including physical ex-
aminations, electrocardiograms, chest X rays, and echocardiograms were per-
formed in order to classify patients into different groups as previously defined by
us (43). Clinical groups were assigned as follows: the I group (n ? 7), consisting
of patients who did not present with any clinical manifestations or alterations
upon clinical, radiological, and echocardiographic examination; the DC group
(n ? 5), consisting patients who presented with right and/or left ventricular
dilation, global left ventricular dysfunction, and alterations in the cardiac electric
impulse generation and conduction. In the latter group, the alterations were
evident in electrocardiograms, chest X rays, and echocardiography, which
showed the occurrence of heart enlargement in all cardiac patients analyzed. Left
ventricular ejection fraction (LVEF) and left ventricular diastolic diameter
(LVDD) were used as clinical parameters of ventricular function for the Chagas’
patients (44). We also included in our analysis individuals without Chagas’
disease (nonchagasic group [N]; n ? 7), as determined by negative specific
serological tests for Chagas’ disease. Individuals with the digestive form of
Chagas’ disease were not included in this study due to low incidence of well-
documented cases in our geographical location in Brazil. Characteristics of the
study groups are summarized in Table 1. We excluded from our study individuals
with any other chronic inflammatory diseases, valvular heart disease, coronary
artery disease, arterial hypertension, diabetes mellitus, alcoholism, and bacterial
infections. All individuals included in this work were volunteers, and treatment
and clinical care were offered to all patients, as needed, despite their enrollment
in this research project. This study is part of an extended project evaluating risk
factors for cardiac damage/involvement in Chagas’ disease, which has the ap-
proval of the Research Ethics Committee of the Federal University of Minas
Gerais (COEP-UFMG-ETIC006/05) and is in accordance with the Helsinki
Declaration. Peripheral blood was collected by venipuncture, and informed con-
sent was obtained from all individuals.
Parasites. Trypomastigotes of the Y strain of T. cruzi were grown in Vero or
L929 cell lines, as previously performed by us (49). Briefly, cells were infected
with 10 trypomastigotes/cell and, after free trypomastigotes were removed by
washing with culture medium, were maintained in RPMI medium enriched with
5% fetal calf serum and antibiotics (penicillin, 500 U/ml; streptomycin, 0.5
mg/ml) for approximately 5 days. After this period, trypomastigotes ruptured the
cells and were collected from the supernatant. The contamination with amasti-
gote forms was always below 3%. Parasites obtained in such a manner were used
for infecting blood cells from patients and nonchagasic individuals.
Infection of blood cells from patients and nonchagasic individuals with T.
cruzi trypomastigotes. Infection of peripheral blood cells was performed using
10 trypomastigotes/cell, as previously described (49). Briefly, cells and parasites
were incubated at 37°C in 5% CO2for a period of 3 h. After this time, cells were
washed by centrifugation with phosphate-buffered saline (PBS) for removal of
free trypomastigotes. After centrifugation, the supernatant was removed, and a
4422VILLANI ET AL. INFECT. IMMUN.

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volume of RPMI medium supplemented with antibiotic/antimycotic (amphoter-
icin B, 0.25 ?g/ml; penicillin, 200 U/ml; and streptomycin, 0.1 mg/ml) and L-
glutamine (1 mM) equal to the amount of blood initially incubated was added to
the tubes. Infected cells were incubated at 37°C in 5% CO2for a period of 14 h.
After this period, brefeldin A (1 ?g/ml) was added to prevent protein secretion,
and cultures were reincubated for an additional 4 h. For all individuals, we
carried out cultures of blood submitted to the same procedures described above,
but in the absence of parasites, as nonstimulated controls.
Determination of the frequencies of DN T cells and expression of cytokines by
?? and ?? DN T cells. Frequencies of ?? and ?? DN T cells, as well as
expression of IFN-?, TNF-?, IL-17, and IL-10 by these DN T-cell subpopula-
tions, were determined by flow cytometry. Infected cells (treated as described
above) or noninfected blood cells were harvested after the final 18 h of culture
and submitted to specific staining for the above-mentioned molecules. We used
a combination of CyChrome-labeled anti-CD4 and -CD8 to detect DN T cells, as
previously done by us (4). Fluorescein isothiocyanate (FITC)-labeled anti-?? or
anti-?? T cells were also used in the staining to identify the specific subpopula-
tions. Cells were harvested and plated at a concentration of 200,000 cells/well and
incubated with a 20-?l mixture of the surface antibodies (anti-CD4?anti-CD8?
labeled with CyChrome and anti-?? or anti-?? labeled with FITC) for 15 min at
4°C. Samples were washed three times in phosphate-buffered saline (PBS)–1%
bovine serum albumin (BSA) and fixed by a 20-min incubation with a 2%
formaldehyde solution. After the fixing solution was removed by centrifugation
and cells were washed with PBS, we permeabilized the cells by incubation for 10
min with a 0.5% saponin solution and proceeded with intracellular cytokine
labeling. Samples were incubated with phycoerythrin (PE)-labeled anticytokine
monoclonal antibodies for 20 min at room temperature, washed twice with 0.5%
saponin solution, resuspended in PBS, and read in a flow cytometer. A minimum
of 40,000 gated events from each sample were collected and analyzed using the
FlowJo program. Analysis was performed by gating on the lymphocyte popula-
tion and further gating on the CD4?CD8???- or ??-producing T cells to
determine the expression of the different molecules, as previously done by us (4).
Statistical analysis. ThemeansofthedifferentgroupswerecomparedusingTukey-
Kramerall-paircomparisonanalysisofvariancecontainedwithintheJMPsoftwarefrom
SAS.Apairedttestwasusedtoascertaindifferencesamongnoninfectedversusinfected
cultureswithinthesamegroupofpatients.CorrelationanalysiswasdoneusingPearson’s
correlation coefficient. Differences that returned P values of less than or equal to 0.05
were considered statistically significant from one another.
RESULTS
In vitro infection with trypomastigote forms of T. cruzi in-
duces an expansion of ?? and ?? DN T cells from chronic
Chagas’ disease patients. To determine the frequency of DN
T-cell subsets in chronic Chagas’ patients and nonchagasic
individuals, we performed flow cytometric analysis of periph-
eral blood cells from these individuals, as described above. The
analyses were carried out using nonstimulated cells to provide
information about the frequency of these cells ex vivo from the
patients, as well as after in vitro infection with trypomastigote
forms of T. cruzi, to determine whether contact with the par-
asite led to the expansion of these cells and, if so, to what
extent in the different groups. Our analysis showed that the
frequencies of ?? and ?? DN T cells in nonstimulated cultures
were similar among groups (Fig. 1 A, white bars). Moreover,
we observed that within the total DN T-cell population, the
frequencies of ?? and ?? TCR-expressing T-cell subpopula-
tions were similar among groups (means ? standard deviations
for ?? TCR subpopulations were 23% ? 8% [N], 30% ? 11%
[I], and 25% ? 7% [DC], while those for ?? TCR subpopula-
tions were 77% ? 8% [N], 70% ? 11% [I], and 75% ? 7%
[DC]). Exposure of the cells from indeterminate and cardiac
patients, as well as nonchagasic individuals to trypomastigote
forms of T. cruzi led to an expansion of ?? and ?? DN T cells
in all groups (Fig. 1A, dark bars; representative fluorescence-
activated cell sorting [FACS] plots are shown in B).
?? and ?? DN T cells from chagasic patients display dis-
tinct immunoregulatory profiles. In order to determine the
functional characteristics of ?? and ?? DN T cells from pa-
tients with polar clinical forms of Chagas’ disease, we investi-
gated the expression of inflammatory (IFN-?, TNF-?, and IL-
17) and anti-inflammatory (IL-10) cytokines by these cells in
nonexposed cultures and cultures exposed to the parasite. We
observed that, whereas there was no difference in the frequen-
cies of ?? and ?? DN T cells expressing any of the cytokines in
nonstimulated cultures of the different groups (Fig. 2 and 3,
white bars), exposure to the parasite revealed dramatic differ-
ences among them. In contrast, stimulation of peripheral blood
cells with T. cruzi led to a significant increase in the frequency
of ?? DN T cells expressing IFN-?, TNF-?, and IL-17 from
chagasic patients but not from noninfected individuals (Fig. 2,
left panels). The induction of inflammatory cytokines was more
evident in cells from DC patients exposed to the parasite than
from I patients, as demonstrated by the significantly higher
frequency of cytokine-producing cells in the DC group than in
the N group for all cytokines (Fig. 2, left panels). When the
expression of inflammatory cytokines within the ?? DN T-cell
population was analyzed, T. cruzi-induced increases in cells
expressing IFN-?, TNF-?, and IL-17 were seen for both clin-
ical forms but not for nonchagasic individuals (Fig. 2, right
panels). In this subpopulation, the induction of inflammatory
cytokines was significantly higher in cells from the I and DC
groups than from the N group after exposure to the parasite
(Fig. 2, right panels).
Analysis of expression of the down-modulatory cytokine
IL-10 within the ?? and ?? DN T-cell populations showed that
this cytokine was dramatically induced in T. cruzi cultures with
cells from indeterminate patients (Fig. 3A; a representative
FACS plot is shown in B). This increase in the frequency of
IL-10-producing cells from indeterminate patients was seen
when unstimulated and T. cruzi-stimulated cultures were com-
pared, as well as in comparisons of T. cruzi-stimulated cultures
of the I group versus the N and DC groups within the ?? DN
T-cell subpopulation (Fig. 3). To determine the regulatory
TABLE 1. Individuals analyzed in the study and their clinical status
Patient
no.
Serology
for
Chagas’
disease
Clinical form
Age
(yr)
Sex
LVEF
(%)
LVDD
(mm)
N1
N2
N3
N4
N5
N6
N7
I1
I2
I3
I4
I5
I6
I7
DC1
DC2
DC3
DC4
DC5
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
43
27
23
31
21
19
20
55
38
50
34
68
39
38
59
53
ND
50
63
Female
Female
Female
Female
Male
Male
Male
Male
Female
Female
Female
Female
Female
Female
Female
Male
Male
Male
Male
Indeterminate
Indeterminate
Indeterminate
Indeterminate
Indeterminate
Indeterminate
Indeterminate
Cardiac
Cardiac
Cardiac
Cardiac
Cardiac
60
70
68
67
NDa
69
66
34
22
ND
51
37
55
52
50
46
ND
46
49
70
69
ND
64
65
aND, not determined.
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potentials of each subpopulation of ?? and ?? DN T cells from
I and DC patients, we calculated regulatory ratios by dividing
the frequency of cells producing IL-10 by the frequency of cells
producing either IFN-?, TNF-?, or IL-17. These results show
that ?? DN T cells from group I have a much greater down-
modulatory profile than ?? DN T cells from DC patients (Ta-
ble 2). An analysis of the relative contribution by DN and other
T cells to the overall frequency of IL-10-producing lympho-
cytes demonstrates that, on average, DN T cells account for at
least 32% of the total IL-10 expression. This is a striking
contribution, given that DN T cells represent a minority pop-
ulation of T cells. Interestingly, while the contribution of DN T
cells to the overall IL-10 expression by lymphocytes in non-
stimulated cultures from nonchagasic individuals was lower
than that of CD4?CD8?T cells (33% ? 8% versus 51% ?
13%; P ? 0.05), it was higher in nonstimulated cultures from
indeterminate patients (49% ? 22% versus 27% ? 12%; P ?
0.05). No statistically significant changes were observed in
comparisons of the DN T cells and CD4?CD8?T cells from
cardiac patients.
Higher frequencies of IL-10-producing ?? DN T cells are
correlated with improved clinical parameters of heart function
in human Chagas’ disease. Previous studies performed by our
group have suggested that IL-10 has a protective role in human
Chagas’ disease through an association between the indeter-
minate clinical form of Chagas’ disease and high-producing
genotypes for IL-10 (12). In order to further determine if the
IL-10-producing ?? DN T-cell subpopulation is correlated with
improved cardiac function, and thus with a possible protective
role in human Chagas’ disease, we performed a correlative
analysis between the frequency of these cells and two clinical
parameters of cardiac function: left ventricular ejection frac-
tion (LVEF) and left ventricular diastolic diameter (LVDD).
These distinct clinical parameters are directly and inversely cor-
FIG. 1. T. cruzi activation of peripheral blood cells induces expansion of ?? and ?? double-negative (CD4?CD8?) T cells. Whole blood
cells from noninfected controls (N), indeterminate chagasic patients (I), and dilated cardiac chagasic patients (DC) were incubated overnight
as described in Materials and Methods with either medium alone (MED) or with live T. cruzi parasites (TRP) and then analyzed for the
frequency of ?? and ?? CD4?CD8?T cells using flow cytometry. Panel A shows the average frequencies for each group ? standard
deviations. The numbers of individuals in each group were as follows: N, seven; I, seven; and DC, five. Statistical significance is indicated
in each graph, with differences between groups indicated by common numbers. Comparisons between groups were performed using a
Tukey-Kramer comparison of all pairs, and comparisons within groups (MED versus TRP) were performed using a paired t test, as described
in Materials and Methods. All patients were meticulously classified based on clinical criteria as described in Materials and Methods. Panel
B shows representative dot plots from an indeterminate patient and gating for analysis of ?? and ?? CD4?CD8?T cells. Both anti-CD4 and
anti-CD8 antibodies were conjugated with CyChrome, allowing identification of the DN T cells using specific antibodies against both ?? and
?? T-cell receptors conjugated with FITC. The gates used for determining the percentage of DN T cells and further analysis of cytokine
expression in the DN T-cell populations are shown.
4424VILLANI ET AL.INFECT. IMMUN.

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related with better cardiac function, respectively (44). Strikingly, a
significant positive correlation was seen between higher LVEF
and higher frequencies of IL-10-producing ?? DN T cells (Fig. 4
A). Moreover, a highly significant negative correlation between
lower LVDD and higher frequencies of IL-10-producing ?? DN
T cells was also seen (Fig. 4 A). Interestingly, although ?? DN T
cells also express IL-10 upon stimulation with the parasite, we did
not observe correlations between the frequency of these cell sub-
populations and the clinical parameters analyzed (Fig. 4 B).
These data suggest that IL-10-producing ?? DN T cells display an
important immunoregulatory role that leads to maintenance of
better cardiac function in chagasic patients.
DISCUSSION
Human infection with T. cruzi is the cause of Chagas’ dis-
ease, an illness that currently affects approximately 18 million
people in Latin America, where it is considered endemic. In
addition, it is estimated that 100 million people are at risk of
infection with T. cruzi. Although treatment is available and
relatively effective (40, 47), toxicity and lack of widely distrib-
uted pediatric formulations are still major problems in human
Chagas’ disease. While vector transmission was controlled in
certain areas of South America, disease transmission via blood
transfusion and organ transplant has brought the disease to the
attention of health professionals in Latin America and other
countries where the disease is not endemic, such as the United
States and other countries (28). Moreover, cases of acute Cha-
gas’ disease have been described in areas where acute cases were
not reported for over 15 years (50). Despite the fact that most
Chagas’ patients display a relatively mild, asymptomatic, clinical
form of the disease, about 30% of the patients develop severe
disease, leading to cardiac involvement and, often, death (44).
FIG. 2. T. cruzi activation of peripheral blood cells induces specific inflammatory cytokine production by ?? and ?? DN (CD4?CD8?) T cells
from both indeterminate and dilated cardiac chagasic patients. Whole blood cells from noninfected controls (N), indeterminate chagasic patients
(I), and dilated cardiac chagasic patients (DC) were incubated overnight with either medium alone (MED) or with live T. cruzi parasites (TRP)
and then analyzed for the frequency of ?? or ?? DN T cells producing specific cytokines using flow cytometry, as described in Materials and
Methods. The data represent the average for each group ? standard deviations. The numbers of individuals in each group were as follows: N,
seven; I, seven; and DC, five. The top panel shows the average percentage of IFN-?-producing cells within ?? or ?? DN T cells from individual
cultures without (MED) or with (TRP) stimulus. The middle panel shows the same for TNF-?-producing cells within ?? or ?? DN T cells, and
the bottom panel shows the values for IL-17-producing cells within ?? or ?? DN T cells. Statistical significance is indicated in each graph, with
differences between groups indicated by common numbers. Comparisons between groups were performed using a Tukey-Kramer comparison of
all pairs, and comparisons within groups (MED versus TRP) were performed using a paired t test, as described in Materials and Methods. All
patients were meticulously classified based on clinical criteria, as described in Materials and Methods.
VOL. 78, 2010CD4?CD8?T CELLS IN HUMAN CHAGAS’ DISEASE 4425

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Thus, the social and economic burdens caused by Chagas’ disease
place it among the most morbid of all parasitic diseases.
The mechanisms behind the development of the severe car-
diac form of Chagas’ disease have not been completely eluci-
dated. However, it is well accepted that T cells are key players
in mounting an immune response during the chronic phase of
the disease (17). Thus, T-cell activation and function are crit-
ical in determining the clinical outcome of Chagas’ disease.
Cardiac patients display a highly activated, inflammatory T-cell
response both in situ (13, 24, 41) and in the peripheral blood (2,
16, 19, 32). Interestingly, however, patients who do not develop
pathology and remain asymptomatic also display a high fre-
quency of activated T cells in their bloodstream (18). This
apparent contradiction has been better understood more re-
cently, mainly due to the use of two important approaches: (i)
clear definition of patient clinical forms by performing refined
clinical analysis and (ii) identification and characterization of
T-cell subpopulations that display distinct functional activities.
Thus, recent studies using patients with well-defined clinical
forms have shown that although T-cell activation is observed in
severe and asymptomatic Chagas’ patients, these cells have
distinct functional potentials (17). Most studies have focused
FIG. 3. ?? DN (CD4?CD8?) T cells from indeterminate chagasic patients display a biased down-modulatory profile following stimulation with
T. cruzi. Whole blood cells from noninfected controls (N), indeterminate chagasic patients (I), and dilated cardiac chagasic patients (DC) were
incubated overnight with either medium alone (MED) or with live T. cruzi (TRP) and then analyzed using flow cytometry for the frequency of ??
or ?? DN T cells producing IL-10, as described in Materials and Methods. The data represent the average for each group ? standard deviations.
The numbers of individuals in each group were as follows: N, seven; I, seven; and DC, five. Panel A shows the average percentage of
IL-10-producing cells within the ?? or ?? DN T-cell population from individual cultures without (MED) or with (TRP) stimulus for each group.
Statistical significance is indicated in each graph, with differences between groups indicated by common numbers. Comparisons between groups
were performed using Tukey-Kramer comparison of all pairs, and comparisons within groups (MED versus TRP) were performed using a paired
t test, as described in Materials and Methods. Panel B shows representative dot plots and gating for analysis of ?? and ?? CD4?CD8?T cells
producing IL-10. Both anti-CD4 and anti-CD8 antibodies were conjugated with CyChrome, allowing identification of the DN T cells using specific
antibodies against both ?? and ?? T-cell receptors conjugated with FITC. The gates used for determining the percentage of DN T cells producing
IL-10 were then determined in a histogram using anti-IL-10 conjugated with PE. The percentages of cells producing IL-10 from cultures either
with medium alone or with T. cruzi stimulation were determined, as described in Materials and Methods. All patients were meticulously classified
based on clinical criteria, as described in Materials and Methods.
4426 VILLANI ET AL.INFECT. IMMUN.

Page 7

on the analysis of expression of factors that control the estab-
lishment of inflammatory responses in Chagas’ disease, such as
inflammatory cytokines and chemokines (20, 21). Studies per-
formed by us and other groups have shown that major T-cell
populations, defined by the expression of CD4 and CD8, dis-
play phenotypic and functional differences in individuals with
different clinical forms of Chagas’ disease. To this end, the
frequencies of memory cells, as well as senescent cells, have
been associated with the chronic cardiac form of Chagas’ dis-
ease (1, 2, 23). While these studies have provided critical in-
formation, the determination of the contribution of distinct
subpopulations to the immunoregulation and functional activ-
ities, as well as the antigens that lead to their activation, is
critical for the understanding the mechanisms of generation of
pathogenic versus protective responses in Chagas’ disease.
A quantitatively small subpopulation of T cells that does not
express CD4 or CD8 molecules has been identified, and be-
cause of the ability of these cells to tolerate chronic stimulation
due to the lack of the stabilizing CD4 or CD8 molecules, they
have been shown to be critical in chronic immune diseases,
especially auto-immune processes (8, 30). Furthermore, a large
portion of these cells are activated by recognizing lipid/glyco-
lipid antigens presented via CD1 molecules (36). Glycolipid
determinants from T. cruzi have been shown to be important in
the activation of cellular immune responses in experimental
infection (34). Although previous studies of murine infection
with T. cruzi suggested that CD1 molecules were not critical in
eliciting cellular responses to parasite components (34, 39),
others have shown that CD1 presentation is important for
natural killer T (NKT)-cell activation (14, 15, 31).
The role of DN T cells in T. cruzi infection has not yet been
clarified. It has been shown that mice infected with the parasite
display a 40- to 100-fold increase in the frequency of liver ??
CD4?CD8?lymphocytes, associated with expression of IFN-?
(45). Interestingly, the same group later showed that the liver
is an important organ for parasite clearance in chronic infec-
tion (46). An increase in the DN T-cell frequency in the liver
of animals infected with Plasmodium was also associated with
TABLE 2. Indeterminate patients maintain a regulatory ratio of
IL-10-producing cells
Patient group
Regulatory ratioa
IL-10/IFN-?
IL-10/TNF-?
IL-10/IL-17
Indeterminate
Cardiac
1.01 ? 0.26
0.32 ? 0.07b
1.26 ? 0.21
0.55 ? 0.14b
1.66 ? 0.21
0.89 ? 0.09b
aThe frequency of ?? DN T cells expressing the cytokines of interest following
in vitro stimulation with T. cruzi was determined as described in Materials and
Methods for each patient and then used to calculate regulatory ratios by dividing
the frequency of cells producing IL-10 (a downregulatory cytokine) by the fre-
quency of cells producing IFN-?, TNF-?, or IL-17.
bThe values represent the average of ratios for IL-10/given cytokine ? the
standard error for seven indeterminate and five cardiac patients. In all cases the
comparison between the ratios for the indeterminate versus the cardiac groups
returned a P value of ?0.01.
FIG. 4. Higher frequencies of IL-10-producing ?? DN (CD4?CD8?) T cells are correlated with better heart function in chagasic patients. The
frequency of ?? DN T cells producing IL-10 following stimulation with T. cruzi was calculated from a group of chagasic patients who had associated
detailed clinical data measuring ventricular function. These measurements were the left ventricular ejection fraction (LVEF) and left ventricular
diastolic diameter (LVDD). The higher the LVEF, the better the ventricular function, and the lower the LVDD, the better the ventricular function.
Panel A shows Pearson’s correlation plots between the frequency of IL-10-producing ?? DN T cells and LVEF or LVDD. Both plots demonstrate
a highly significant correlation between higher frequencies of IL-10-producing ?? DN T cells and better ventricular function. In contrast, in panel
B, no correlation is seen between IL-10-producing ?? DN T cells and measurements of ventricular function. Clinical data for a total of nine
chagasic patients were used in this analysis. Statistical significance (P value) is indicated in each graph together with the r2value.
VOL. 78, 2010CD4?CD8?T CELLS IN HUMAN CHAGAS’ DISEASE 4427

Page 8

parasite inhibition (35). Infection of rats with the highly viru-
lent CL-Brenner clone of T. cruzi was associated with an ex-
pansion of CD4?CD8?T cells and IFN-? production (33).
Recent studies have also pointed to important roles of DN T
cells in human parasitic diseases. We have shown that ?? and
?? DN T cells display distinct immunoregulatory profiles in
human cutaneous leishmaniasis (4, 25). Moreover, a high fre-
quency of DN T cells was observed in the peripheral blood of
individuals with Plasmodium falciparum malaria (52). In this
work, we performed an analysis of the frequency of DN T cell
?? and ?? subpopulations in individuals with polar clinical
forms of Chagas’ disease. Our results showed that although
there were no quantitative differences in the frequencies of
these cells freshly isolated from chagasic patients and nonin-
fected individuals, T. cruzi infection led to an expansion of DN
T cells in vitro, and these cells were quite different in their
immunoregulatory potentials. Although a parasite-induced ex-
pansion of DN T cells was observed in cultures of cells from
patients as well as from noninfected individuals, the DN T cells
from noninfected individuals did not express parasite-induced
cytokines, compatible with a primary response. On the other
hand, expanded cells from patients produced high levels of
cytokines, indicative of an antigen-specific recall response, and
also showed different cytokine expression profiles in indeter-
minate and cardiac patients. We observed that ?? DN T cells
from individuals of the cardiac clinical form of Chagas’ disease
display higher expression of inflammatory cytokines upon in
vitro stimulation with T. cruzi. Interestingly, ?? DN T cells from
indeterminate patients displayed a markedly high expression of
IL-10 following T. cruzi stimulation, which was not observed in
cardiac patients. Analysis of the ratio IL-10/inflammatory cy-
tokines revealed a clear down-modulatory environment asso-
ciated with ?? DN T cells in indeterminate patients and not in
cardiac patients. Given that we do not know the exact nature of
the antigen responsible for the activation of these cells, we
have not yet focused on any specific DN T-cell subpopulation,
such as the DN NKT cells. Further studies are being carried
out in our laboratory to clarify these questions. However, the
observed functional differences presented here are clearly as-
sociated with important clinical features of the patients and
continue to support earlier findings by our group and others
defining key differences in the immunoregulatory environ-
ments between indeterminate and cardiac chagasic patients
(20).
Monitoring cardiac function is an important procedure that
permits one to follow the course of pathology development and
worsening of human Chagas’ disease. Unfortunately, due to
the high costs of several of the required exams, it is not always
possible to perform these procedures. We evaluated a group of
clinically well-defined Chagas’ patients in which two measures
of cardiac function were performed: left ventricular ejection
fraction and left ventricular diastolic diameter. These clinical
characteristics, although physiologically related, reflect differ-
ent levels of cardiac lesion. The greater the LVEF and the
smaller the LVDD, the better the cardiac function. A positive
correlation between a higher frequency of IL-10-producing ??
DN T cells and improved cardiac function as measured by
LVEF was seen. Moreover, the higher the frequency of IL-10-
producing ?? DN T cells, the lower the LVDD, which again
indicates the association of IL-10-producing ?? DN T cells with
better cardiac function. Previous studies performed by us
showed that a down-modulatory profile, as accessed mainly by
IL-10 and CTLA-4 expression, was predominant in indetermi-
nate patients (48, 49). Moreover, we demonstrated that IL-10
promoter gene polymorphism, which leads to high IL-10 ex-
pression, is associated with the occurrence of the indetermi-
nate clinical form. Here, we suggest that IL-10 derived from ??
DN T cells may also be involved in protection. This is an
important finding since these cells are likely activated via dis-
tinct mechanisms compared to the other cell populations stud-
ied to date. This could aid in the development of novel antigen-
based prophylactic or therapeutic interventions.
An important question still unanswered is why these cell
populations display distinct functional capabilities in patients
with indeterminate and cardiac clinical forms. This is particu-
larly intriguing when we remember that indeterminate pa-
tients, who apparently display a modulated response that may
be important for avoiding tissue inflammation, may develop
cardiac disease in the future. The hypothesis is that these
individuals undergo cellular functional changes, which would
lead to pathology establishment. Assuming that these changes
are a cause and not a consequence of pathology, then identi-
fying such differences and determining their causes will provide
critical information for preventing cardiac damage and a wors-
ening clinical pathology.
ACKNOWLEDGMENTS
This investigation received financial support from the UNDP/
World Bank/WHO Special Programme for Research and Training
in Tropical Disease, CNPq Universal grant, FAPEMIG, FINEP
CT-Infra, and CNPq/Ministe ´rio da Sau ´de INCT-DT. W.O.D.,
M.O.D.C.R., L.R.D.V.A., L.M.D.M., and K.J.G. are CNPq fellows;
F.N.A.V. and J.S.C.D.S. are CAPES fellows.
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