Immunology and Cell Biology (2009) 87, 149–153 & 2009 Australasian Society for Immunology Inc. All rights reserved 0818-9641/09 $32.00 www.nature.com/icb
ORIGINAL ARTICLE
Natural CD4+ T-cell responses against Trypanosoma cruzi KMP-11 protein in chronic chagasic patients Adriana Cuellar1, Francia Rojas1, Natalia Bolan˜os2, Hugo Diez3, Marı´a del Carmen Thomas4, Fernando Rosas5, Victor Velasco5, Manuel Carlos Lo´pez4, John Mario Gonza´lez2 and Concepcio´n Puerta3 Trypanosoma cruzi kinetoplastid membrane protein-11 (KMP-11) is able to induce protective immunity in mice. In humans, T-cell immunity during Chagas disease has been documented using parasite antigens allowing the identification of specific CD8+ T cells. However, little is known about the CD4+ T-cell response during the evolution of the disease. In this paper, the induction of a natural CD4+ T-cell response against the KMP-11 protein in T. cruzi infected humans was studied to assess whether this parasite-derived protein could be processed, presented and detected as a major histocompatibility complex class II restricted epitope. The results show that helper T cells from 5 out of 13 chagasic patients specifically produced interferon-g after exposure to the KMP-11 antigen, whereas healthy donors and non-chagasic cardiopathic patients did not respond. This is the first description of T. cruzi KMP-11 protein recognition by CD4+ T cells in chronic chagasic patients. Immunology and Cell Biology (2009) 87, 149–153; doi:10.1038/icb.2008.76; published online 28 October 2008 Keywords: CD4+ T lymphocytes; Chagas disease; immune response; Trypanosoma cruzi
Trypanosoma cruzi, a human hemoflagellate parasite, causes the Chagas disease, which affects 15 million people in 21 endemic countries with an annual incidence of 41 200 cases around the world.1 In Colombia, it is estimated that 700 000 people are infected and 23% of the population is at risk of contracting the disease with 30 000–40 000 new cases per year.2 Chagas disease has a natural disease history that includes three different phases. The acute onset is solely seen in endemic areas with some clinical characteristics not often detected. Most people recovered from this phase and moved to the indeterminate phase where parasites persist in blood with no symptoms for at least 20 or 30 years. Some infected individuals develop a chronic disease indicating that their immune response is not capable of controlling the infection. This parasite persistence affects tissues, giving rise to local inflammatory lesions that are characteristic of the chronic phase with heart or visceral enlargement. Heart involvement could progress to a marked cardiomegaly, cardiac failure, severe arrhythmia and death.3,4 The immune response plays a key role in controlling the infection but has also been implicated in the pathogenesis of the chronic disease. Several studies have reported that the kinetoplastid membrane protein-11 (KMP-11) is able to elicit both B- and T-cell lymphoproliferation as well as cytotoxic responses.5–10 Indeed, Maran˜on et al.6
showed that the immunization of HLA-A2/Kb transgenic mice with the recombinant fusion protein encompassing the T. cruzi heat-shock protein of 70 kDa (HSP70) and KMP-11 sequences induces a cytotoxic response against human cells expressing the KMP-11 protein. An immunodominant HLA-A*0201 restricted cytotoxic epitope, termed K1, was identified and located between amino acids 4–12 of the KMP11 protein. Moreover, mice immunized with the chimerical gene that codifies for the KMP-11 and HSP70 proteins produced antibodies and CTL response that induced protection after parasite challenge.8 Earlier, it was shown in our group that CD8+ T cells from chagasic patients recognized the K1 peptide in the context of the HLA-A*0201 and specifically secreted interferon-g (IFNg), suggesting that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of the Chagas disease.11 In contrast, information about specific CD4+ T cells in infected humans is scarce. On the basis of previous results in the murine model, it is clear that the coordinated activation of both CD4+ and CD8+ T cells are essential for infection control.12 The aim of this study is to identify whether the KMP-11 protein has the potential to enter in the major histocompatibility complex class II processing and presentation pathway to further characterize the CD4+ T-cells response during the infection. The presence of IFNg or interleukin
1Grupo de Inmunobiologı´a y Biologı´a Celular, Departamento de Microbiologı´a, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogota´, Colombia; 2Grupo de Ciencias Ba´sicas Me´dicas, Universidad de los Andes, Bogota´, Colombia; 3Laboratorio de Parasitologı´a Molecular, Departamento de Microbiologı´a, Pontificia Universidad Javeriana, Bogota´, Colombia; 4Departamento de Biologı´a Molecular, Instituto de Parasitologı´a y Biomedicina Lo´pez Neyra, CSIC, Granada, Spain and 5Fundacio´n Clı´nica Abood Shaio, Bogota´, Colombia Correspondence: Dr C Puerta, Laboratorio de Parasitologı´a Molecular, Departamento de Microbiologı´a, Facultad de Ciencias, Pontificia Universidad Javeriana, Carrera 7a No 43-82, Edificio 52, Oficina 608, Bogota´, Distrito capital 1, Colombia. E-mail:
[email protected] Received 10 January 2008; revised 18 September 2008; accepted 21 September 2008; published online 28 October 2008
CD4+ T-cell responses against T. cruzi KMP-11 protien A Cuellar et al 150
(IL)-4-secreting CD4+ T-cells specific for the KMP-11 protein was determined in cells from infected individuals, healthy controls and non-chagasic cardiopathic patients.
encompassing the CD3+ CD4+ population combined with the expression of CD69 in cells from chagasic patients (Figure 2a). In all donor groups, the majority of cytokine-secreting cells also expressed the activation marker after stimulation with polyclonal stimulus (Figure 2b). Neither IFNg nor IL-4 was detected in non-stimulated cells derived from blood samples of all kind of donors. Interestingly, CD4+ T-cells derived from chagasic patients showed lower percentages of IFNg-secreting cells after polyclonal stimulus compared with controls of healthy donors with significant statistical differences of P¼0.03. Moreover, no differences were observed in this response between indeterminate and chronic chagasic patients (Figure 2c). In addition, when secretion of IL-4 was compared after stimulation with SEB, no differences were found among any group of donors (Figure 2c). Analysis of CD4+ T cells from chagasic patients showed that 5 out 13 had specific IFNg production after co-culturing with the KMP-11 protein (Table 1). Among the responders, one was G0, one G2 and three G3, according to the Kuschnir13 classification in which G0 refers to patients with normal findings on electrocardiograms, transthoracic echocardiograms and chest radiographs; G2, patients with abnormal findings on electrocardiograms, and findings of cardiomegaly in both transthoracic echocardiograms and chest radiographs; and G3, patients with abnormal findings on electrocardiograms, findings of cardiomegaly in both transthoracic echocardiograms and chest radiographs, and clinical evidence of heart failure. Therefore, KMP-11 responders are mostly G2–G3 patients who exhibit more severe clinical symptoms. An example of one KMP-11 responder chagasic donor (G3), whose CD4+ T cells specifically secreted IFNg, is shown in Figure 3a. Furthermore, the IFNg response observed in the chagasic donors is dependent on the KMP-11 protein concentration. Thus, using the KMP-11 protein at 10 mg ml1 induced the highest percentage of antigen-specific CD4+ T-cells response to the parasite antigen as shown in Figure 3b. In contrast, CD4+ T cells from healthy donors and non-chagasic cardiopathic patients, in spite of exhibiting a similar behavior after treatment with polyclonal stimulus (data not shown),
RESULTS First, to assess cellular status against different stimuli, surface CD69 expression was determined as a cell activation marker. The results showed that expression of CD69 was similar in healthy controls and chagasic patients in non-stimulated cultures or after stimulation with Staphylococcus aureus enterotoxin B (SEB) used as a polyclonal stimulant (Figure 1). To determine the Th1/Th2 profile of cytokine secretion after stimulation, a flow cytometry analysis was performed in the region CD3+ CD4+
% CD69 positive cells
60 40 20 6 4 2 0 NS
SEB
KMP-11 10µg
KMP-11 1µg
Figure 1 Expression of the activation marker CD69 in CD3+ CD4+ T cells in cultures non-stimulated (NS), stimulated with the Staphylococcus aureus enterotoxin B (SEB) or kinetoplastid membrane protein-11 (KMP-11) protein (1 or 10 mg ml1), in healthy donors (empty bars) and chagasic patients (gray bars). The results are shown as the average of the percentages of positive cells and respective standard error (s.e.m.).
SEB Healthy controls Indeterminate chagasic patients Chronic chagasic patients
CD3
NS
IFNγ
CD4
% cytokine secreting cells
15
10
*
*
5
0 IL-4
CD69+ IFNγ+
CD69+ IL-4+
CD69 Figure 2 Response of CD3+ CD4+ CD69+ T-cells non-stimulated (NS) or co-cultured with polyclonal stimulation (Staphylococcus aureus enterotoxin B (SEB)). Dot plot analysis representative of 1 out of 21 studied subjects showing the phenotype distribution (a) and cytokine secretion (b). Comparison of cytokine secretion in healthy donors (empty bars), indeterminate chagasic patients (gray bar) and chronic chagasic patients (black bars) (c). The results are shown as the average of the percentages of positive cells and respective standard error (s.e.m.). *Denotes statistical differences, P¼0.03. Immunology and Cell Biology
CD4+ T-cell responses against T. cruzi KMP-11 protien A Cuellar et al 151
KMP-11 10 µg/ml
Table 1 Characteristics of the population studied and percentage of interferon-c (IFNc) secreted in reactive chagasic donors Age (years)
Chagas risk factors a
26
M
46
No
Healthy donor
34 48
M M
24 24
No No
Healthy donor Healthy donor
223 224
M F
27 44
No No
Healthy donor Healthy donor
1 2
F M
64 60
Yes Yes
G2 G1
CD4+ IFNγ+
Gender
Clinical condition
CD69 0.8
(0.54)b
3 4
M M
65 60
Yes Yes
G3 G1
5 7
F F
64 60
Yes Yes
G1 G1
8 9
F M
68 37
Yes Yes
G3 (0.37)b G0
25 39
M F
61 67
Yes Yes
G0 G2 (0.26)b (0.55)b
55 57
M F
41 34
Yes Yes
G0 G0
75
F
51
Yes
G2 (0.34)b
45 0
F M
74 70
No No
NCC NCC
Seropositive chagasic patients were classified according to the Kuschnir grading system13 as follows: G0, patients with normal findings on electrocardiograms (ECGs), transthoracic echocardiograms (TTEs), and chest radiographs; G1 patients with normal findings on TTEs and chest radiographs but abnormal findings on ECGs; G2, patients with abnormal findings on ECGs, and findings of cardiomegaly in both TTEs and chest radiographs; G3 patients with abnormal findings on ECGs, findings of cardiomegaly in both TTEs and chest radiographs, and clinical evidence of heart failure. NCC: patients with cardiopathy of non-chagasic origin. Gender, male (M) or female (F) and patient’ age are indicated. aRecognition of at least two of the following findings: presence of the vector, Chagas disease in others family members or living in endemic area. bPercentage of IFNg-secreting CD3+ CD4+ T cells from responder chagasic donors to kinetoplastid membrane protein 11 recombinant protein at 10 mg ml1.
% cytokine secreting cells
Subject code
0.54
0.6
Non-infected donors (NI) Non-responder chagasic donors (nrCh) Responder chagasic donors (rCh)
0.4 0.2 0 KMP-11 10 µg
KMP-11 10 µg
KMP-11 1 µg
KMP-11 10 µg
Figure 3 Specific response of CD3+ CD4+ CD69+ T-cells co-cultured with the kinetoplastid membrane protein-11 (KMP-11) protein. Dot plot analysis representative of 1 out of 13 chagasic patients showing the cells specifically secreting cytokines in response to the KMP-11 protein (a). Percentage of interferon-g (IFNg)-secreting CD3+ CD4+ T cells of seven non-infected donors (NI) and six non-responder chagasic patients (nrCh) against the KMP-11 protein (10 mg ml1), and five responder chagasic donors (rCh) against two different KMP-11 protein concentrations (b). The NI group includes five healthy donors and two individuals with non-chagasic cardiopathy; nrCh group contains one indeterminate and four chronic patients. The results are shown as the average of the percentages of positive cells and respective s.e.m.
Table 2 Stimulation indexa from healthy and chronic chagasic patients against amastigote total lysate
did not respond to the KMP-11 protein either at 1 or 10 mg ml1. In addition, proliferation assays were carried out stimulating peripheral blood mononuclear cells (PBMCs) from four chronic chagasic patients and two healthy donors with both whole parasite lysate from amastigotes and KMP-11 protein. As shown in Table 2, the PBMCs from three patients responded to the KMP-11 recombinant protein, although one of them did not show a proliferative response against amastigotes total proteins. Moreover, as expected, healthy donors did not respond either to the KMP-11 protein or to the total parasite proteins. DISCUSSION The function of CD4+ T cells during the T. cruzi infection in the mouse model has been determined as a reflection of the CD8+ T-cells response. The kinetic of CD8+ T cells is dependent on the parasite dose14,15 and their expansion phase, which peaks after 14 and 24 days post-infection,16 requires the presence of CD4+ T cells.15 In major histocompatibility complex class II-deficient mice, there is a low level of specific CD8+ T cells, which are not sufficient to control the infection in the absence of helper T cells.17 Dissecting the role of the CD4+ T-cells population during T. cruzi infection in the mouse model has shown that infection resistance is dependent on Th1 response.18–20 Decrease of IFNg secretion is associated with susceptibility to the infection21 and control of parasitemia relied on CD4+ T cells that produced Th1-associated cytokines.22 Indeed, protected mice were shown to have a higher level of antigen-specific IFNg-secreting cells during infection23 or after vacci-
Subject code
Amastigote total lysate
KMP-11 protein
BCC1 BCC2
3.0 7.5
6.8 6.7
BCC3
1.3
3.5
BCC4 BHD1
0.1 0.3
1.0 2.4
BHD2
0.1
1.8
Abbreviations: BCC, Bolivian chronic chagasic patients; BHD, Bolivian healthy donor. aStimulation index¼(cpm (stimulated culture)cpm (negative control culture))/cpm (negative control culture).
nation with parasite antigens than non-protected animals.24 In addition, generation of Th1 response during T. cruzi infection seems to be protective in humans. Thus, chronic chagasic patients had lower levels of antigen-specific CD8 T+ cells-secreting IFNg compared with nonsymptomatic individuals.25 Similarly, in congenital disease, it was shown that pregnant T. cruzi-infected women who transmitted parasites to their fetus had decreased production of parasite-specific IFNg in PBMC.26 This trend persisted after delivery, compared with the women who did not transmit parasites to their fetus.26 The results shown here suggest that cells from both chagasic and healthy donors are equally competent in the expression of the CD69 activation marker as shown by the polyclonal stimulation with SEB. However, interestingly, the percentage of activated T-cells expressing IFNg was lower in chagasic patients (without any difference between Immunology and Cell Biology
CD4+ T-cell responses against T. cruzi KMP-11 protien A Cuellar et al 152
indeterminate and chronic patients) than in healthy donors under the same culture condition. The low number of antigen-specific cells detected in some studies in Chagas diseases has been attributed to the decrease in the number of circulating lymphocytes, decreased expression of markers,27 loss of activated cells owing to apoptosis28 or polymorphisms in T-cellepitopes.29 In this perspective, T. cruzi-induced immune suppression would account for some difficulties in tracking CD4+ T-cell response in humans.27,28,30 However, when complete parasite antigen was used to stimulate cells from chagasic patients, the percentages of specific IFNg production by CD4+ T cells ranged from 0.014 to 1.57%,25 but individual parasite antigens were not useful in identifying helper T-cell response. In humans, two previous studies have shown recognition of native antigen from T. cruzi by CD4+ T cells. In one study, parasiteinfected macrophages stimulated CD4+ T cells to secrete cytokines, but no parasite epitopes were identified.31 A second study determined specific recognition of cruzipain epitope in human-derived T-cell clones.32 In this paper, it is shown that 5 out of 13 analyzed chagasic patients had circulating precursor cells with the capability of inducing IFNg in the presence of the KMP-11 protein. Indeed, the fact that PBMCs from healthy donors did not proliferate to the whole parasite lysate or the KMP-11 recombinant protein, even though all PBMCs from patients had responded against the KMP-11 protein, confirmed the specificity of this response. To our knowledge, this is the first description of an ex vivo recognition of a T. cruzi protein by human CD4+ T cells. Thus, a specific response of CD4+ T cells is shown with the production of IFNg cytokine in indeterminate as well as in chronic patients after PBMC stimulation with the KMP-11 recombinant antigen. In patients with severe cardiomiopathy (G2–G3), we found four responders out of five donors. In contrast, in the group of people at the earlier phase of the disease (G0–G1), we found only one responder out of eight donors. These results suggest that the KMP-11 protein could be useful in the study and follow-up of the kinetic of T. cruzi-specific CD4+ T cells associated with the disease severity degree. Interestingly, it was also shown in a mouse model that antigen persistence is important for maintaining a pool of antigen-specific responder cells.33 Recently, it was shown that the exposure of lymphocytes to monocytes infected with T. cruzi induces a higher expression of IFNg in cells from chagasic patients than in cells from non-infected individuals.34 However, the authors did not find differences between lymphocyte response from indeterminate and cardiac patients,34 suggesting that the capacity of cytokine secretion by these cells was not related to disease progression. Currently, a higher number of individuals in all stages of Chagas disease are being analyzed to determine the dynamics of KMP-11 CD4+ T-cell responses. METHODS Selection of study population With the aim of identifying markers that allow the determination of the frequency and role of antigen-specific CD4+ T cells during T. cruzi infection, PBMCs from T. cruzi-infected and non-infected control individuals were obtained. A total of 13 chagasic patients with a different disease severity degree according to the Kuschnir13 classification (Table 1) were tested in this study. Chagasic patients, six males and seven females with an average age of 56±11 years old, were seropositive in both immunofluorescence and enzyme-linked immunosorbent assay tests against T. cruzi parasite, as recommended by WHO guidelines.35 Clinical manifestations of Chagas disease were identified by a detailed clinical examination, electrocardiography and chest X-rays. PBMCs from five healthy donors, four males and one female, and two patients with cardiopathy of non-chagasic origin, one female and one male, who were Immunology and Cell Biology
negative for T. cruzi serological tests, with an average age of 44±21 were also included as controls. The inclusion of all subjects in our investigation was approved by the Ethic Committees of the Pontificia Universidad Javeriana and the Fundacio´n Clı´nica Abood Shaio. Signed informed consents were obtained from all volunteers before inclusion in this study. To assess the specificity of KMP-11 protein response, PBMCs from four Bolivian chagasic patients and two Bolivian healthy donors were purified. This analysis was carried out with the consent of the participants and was approved by the Ethics Committee of the Consejo Superior de Investigaciones Cientı´ficas (Spain).
T. cruzi rKMP-11 protein For the cloning of the T. cruzi KMP11 complete protein, the cDNA corresponding to the T. cruzi KMP11 gene was digested with the MscI and RsaI enzymes, subcloned in the SmaI-digested pQE31 expression vector (Qiagen, Hilden, Germany) and purified as described earlier.10 This recombinant protein was dissolved in dimethylsulfoxide and stored at 70 1C.
Mononuclear cells isolation and lymphocyte stimulation Approximately 10 ml of blood was obtained from patients and non-infected donors by venipuncture into heparinized tubes (Vacutainer, Becton-Dickinson, BD Biosciences, Franklin Lakes, NJ, USA). PBMCs were prepared using ficollhypaque density gradient (Sigma, St Louis, MO, USA) and adjusted to 2106 cells in a final volume of 2 ml of RPMI 1640 medium containing antibiotics, non-essential amino acids, sodium pyruvate and 10% fetal calf serum. PBMCs were incubated with 1 mg 1ml each of anti-CD28 and anti-CD49d and with 1 or 10 mg ml1 or 0.5 mg ml1 of T. cruzi rKMP-11 protein. Staphylococcal enterotoxin B (SEB) 3.7 mg ml1, a policlonal stimulant, was used as positive control.36 In each experiment, non-stimulated cells were used as negative control. The cultures were incubated for 3 h at 37 1C in 5% CO2, followed by an additional 9 h of incubation in the presence of 10 mg ml1 of Brefeldin A (Sigma), to block the secretion of cytokines from the cells. Surface staining was done with anti-CD3-FITC, anti-CD4-PerCP, anti-CD69-APC (BD Pharmingen, San Diego, CA, USA) and, after fixation and permeabilization, the cells were stained with IFNg-PE and IL-4-PE (BD Pharmingen). Isotype-matched antibodies were used as a control of fluorescence. Data were acquired and analyzed using a FACSCalibur flow cytometer (BD Immunocytometry Systems, BD Biosciences, Franklin Lakes, NJ, USA) and the Cell Quest program. At least 50 000 cells were read in the CD3+ CD4+ gate. Percentages of specific cytokine secretion are presented as net values obtained by discounting the background (non-stimulated cells) from the experimental conditions (SEB or KMP-11 protein).
Lymphoproliferation assays Peripheral blood mononuclear cells (PBMCs) were separated from fresh heparinized blood by Lymphoprep (Axis-Shield PoC AS, Norway) density gradient centrifugation. Erythrocytes were removed after treatment with red blood cell lysing buffer (Sigma). PBMCs were washed twice in phosphatebuffered saline pH 7.4, counted and adjusted to 1107 cell ml1 in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated fetal calf serum. The cell suspensions were dispensed into 96-well sterile plates at a density of 1105 cells per well and stimulated with the KMP-11 recombinant protein (20 mg ml1) or 5106 of disrupted T. cruzi parasites per well. The parasites (amastigotes forms, Y strain) were obtained from infected monolayers of LLC-MK2 cells, resuspended in phosphate-buffered saline 0.5 in the presence of protease inhibitors cocktail (Sigma) at 10%, and the total lysate proteins were obtained by a cellular disruption by nitrogen decompression using a Cell disruption bomb (Parr Instruments Company, Moline, IL, USA). All assays were performed in triplicate wells in the presence of IL-2 at a final concentration of 20 U ml1 in a final volume of 200 ml per well in the abovementioned medium. SEB at 100 ng ml1 was used as a positive control. Plates were incubated at 37 1C in a CO2 atmosphere for 5 days. After the addition of [methyl-3H] thymidine (0.5 mCi per well), the cells were incubated for another 24 h. The DNA was immobilized in glass fiber filtermats using an Inothech harvester. The 3H incorporation was measured in a Wallac 1450 microbeta counter device (Perkin Elmer, Waltham, MA, USA).
CD4+ T-cell responses against T. cruzi KMP-11 protien A Cuellar et al 153
Statistical analysis The results are shown as the mean and the standard error of the mean. Non-parametric U Mann–Whitney test and Student’s t-test were used for statistical analyses. A significant difference was considered when Po0.05.
ACKNOWLEDGEMENTS This study was supported by The Academy of Science for the Developing World (TWAS) and Vicerrectorı´a Acade´mica, Pontificia Universidad Javeriana, Bogota´, Colombia, Research Grant and project no. 04-039 RG/BIO/LA and 00000827, respectively. MCT and MCL were supported by P06-CTS-02242 Grant from PAI (Junta de Andalucı´a) and RICET-RD06/0021-0014, Spain. We are indebted to Dr RS Nicholls, M Montilla and AC Flo´rez from Laboratorio de Parasitologı´a, Instituto Nacional de Salud for their performance of IFI and ELISA analyses, respectively. We also thank A Lopez-Barajas for technical assistance in the parasite culture proliferation assays.
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Immunology and Cell Biology
