Increased CD4+/CD8+ Double-Positive T Cells in Chronic Chagasic Patients

Background CD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out. Methodology/Principal Findings Seventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1%±0.9) compared with healthy (1.1%±0.5) and non-chagasic cardiomyopathy (1.2%±0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor. Conclusions Chagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can migrate to the heart tissue.


Introduction
Expression of either CD4 or CD8 on mature peripheral CD3+ T cells is considered to be a mutually exclusive event as a result of the thymic selection, reflecting the specific functions of each major T cell subpopulation. Contrary to this conventional dichotomy, circulating CD4+/CD8+ double positive (DP) T cells have been identified in human peripheral blood and represents between 1 and 3% of the total T lymphocytes population [1]. The role of these DP T cells in health and disease is still under investigation.
Based on the intensity of CD4 and CD8 expression by flow cytometry, two subsets of DP T cells have been defined: CD4 dim / CD8 bright and CD4 bright /CD8 dim lymphocytes [3]. Previous studies of their phenotypic characteristics have shown that the majority of these cells have memory phenotype (CD45RO+). They are also more differentiated than mono-positive T cells, based on its low level of expression of CD27, and they frequently produced either intracellular granzyme B or perforin [10,11]. Functional assays showed that during chronic viral infections, DP T cells secrete cytokines, such as IFN-c, in response to cognate class I restricted antigens [10]. All these results suggest that, although small, DP T cells constitute a highly differentiated memory subpopulation acting in the adaptive immune response against infectious agents [1][2][3][4]10,11].
As this cell population is expanded in several viral and immunological mediated chronic diseases, it seems plausible that DP T cells can contribute to the immune response against chronic parasitic infections. The goal of this study was to determine the frequency, phenotype and effector potential of circulating DP T cells in patients chronically infected with T. cruzi, the etiological agent of Chagas disease. This hemoflagellate parasite, which is found in Central and South America, is transmitted by vectors of the Reduviidae family and produces both an acute and a chronic phase [12,13]. Prevalence of Chagas diseases in Colombia is calculated to be 1'300,000, while 3'500,000 people are at risk of contracting the infection, representing a potential public health problem [13,14]. After T. cruzi inoculation, during the acute phase, the parasite extracellular stages are found in peripheral blood and infected individuals develop constitutive symptoms. Activation of the immune system allows parasite control, although it is not completely eliminated. Parasites invade cells such as monocytes/macrophages, dendritic cells, fibroblast and myocardiocytes, shedding their flagella and becoming intracellular [15]. By mechanisms not clearly understood, parasites persist leading to the chronic phase of the disease. Most of the infected individuals will remain asymptomatic for several years, being classified as indeterminate. Those patients are usually detected by routine serological tests in blood banks. Nearly 20% to 30% of the chronic infected patients will develop tissue damage, being Chagasic cardiomyopathy or digestive disease the most common pathologies [12,13]. What mediates the tissue damage in Chagas is not well understood, but some evidence suggests that parasite persistence and dysfunctional cellular immune response could contribute to this process [12,15]. During the infection, antibodies elicited against T. cruzi antigens help to control blood circulating parasite [16,17] and specific CD4+ and CD8+ T cells act against intracellular forms [18,19]. Mice models demonstrated that T cells response seemed to be crucial for parasite control [20]. Interestingly, acute infection with T. cruzi in mice showed a large increase of CD4+/CD8+ DP T cells in their subcutaneous lymph nodes [21]. Given the characteristics of the DP T cells (memory phenotype and presence of cytotoxic granules), it is of special interest to determine their functional characteristics in chronic chagasic patients.

Ethics statement
Research protocols and informed consents were approved by the Ethical Committees of the Universidad de los Andes (039-2009), Pontificia Universidad Javeriana (01-2010) and the Fundación Abood Shaio (134-2010), Bogotá, Colombia, following the national regulations and the Declaration of Helsinki.

Human donors
Forty-one volunteers who signed the informed consent were enrolled in this study and divided into three groups. The first group included 17 chronic chagasic patients with positive immunofluorescence indirect assay (IFI) and ELISA tests. It was composed of 13 females and 4 males with ages ranging from 36 to 67 years old, recruited at the Fundación Abood Shaio (Bogotá, Colombia) or at Instituto Nacional de Salud; and classified according to the Kuschnir grading system [22]. Seven individuals were classified as indeterminate chagasic patients with no significant findings during clinical assessment (G0 or asymptomatic) and 10 as cardiac chronic chagasic patients (symptomatic) graded as follows: 3 with only abnormal ECG results (G1), 5 with abnormal ECG results and cardiac enlargement (G2) and 2 with abnormal ECG results, cardiac enlargement and clinical signs of heart failure (G3). The second group included 12 donors with cardiomyopathy of non-infectious etiology, including 8 females and 4 males with ages ranging from 28 to 80 years old, and whose anti-T. cruzi antibodies were negative. In this group, cardiomyopathy etiology was: ischemic cardiomyopathy (n = 6), systemic arterial hypertension (n = 4) and idiopathic dilated cardiomyopathy (n = 2). This group of patients was recruited at the Department of Cardiology, Hospital Universitario San Ignacio, Bogotá, Colombia. The third group included 12 uninfected donors who never lived in a Chagas endemic area, and with negative IFI and ELISA tests. They were 9 females and 3 males with ages ranging from 34 to 68 years old. The characteristics of the three groups, denoted as chagasic patients (CP), non-chagasic cardiomyopathy (NCC) and healthy controls (HC), including main clinical, electrocardiogram and echocardiography findings, are resumed in Table 1.

Blood sample and cell surface phenotype
Blood samples were obtained from each donor using EDTA vacuntainer tubes. Total blood (100 ml/tube) was stained with anti-CD3 APC (clone SK7), anti-CD4 PerCP (SK3), anti-CD8 PE (RPA-T8), anti-HLA-DR PE-Cy7 (L243) and anti-CD38 FITC (HIT2). All monoclonal antibodies were purchased from BD Bioscience (San Jose, CA, USA). Blood was stained in darkness for 30 minutes at 4uC and incubated with cell lysis buffer (BD Bioscience) for 10 minutes at room temperature. Then, cells were washed twice in PBS 0.01 M pH 7.4 and gently re-suspended. Samples were acquired and analyzed in a FACS Canto II with FACSDiva software (BD Bioscience). At least 5610 4

Author Summary
Chagas disease, produced by the blood parasite Trypanosoma cruzi, is considered a public health problem in Central and South America. Non sterile immunity can be achieved after acute infection. Parasite persistence can induce tissue damage in nearly 20% to 30% of chronically infected individuals. Indeed, chagasic cardiomyopathy is one of the consequences of the chronic infection. Antigen persistence and dysfunctional cellular immune response have been implicated in T. cruzi pathogenesis. Here, a higher frequency of circulating CD4+/CD8+ double positive T cells in chronic chagasic patients is reported as compared with non infected donors, including those with a non-chagasic cardiomyopathy. This cell subset also expressed more activation markers and stored more intracellular perforin. We have previously reported that CD8+ T cells from T. cruzi infected donors recognized the HLA-A*0201 restricted K1-peptide derived from the KMP-11 protein. Here, double positive T cells displayed higher percentages of recognition for the K1 peptide than single CD8+ T cells. These cells produce little IFN-c, but display degranulation activity that was increased in the symptomatic group. Finally, double positive T cells can be localized in the heart tissue from a chronic chagasic donor.

Staining for intracellular markers
Analysis was done with 2610 6 of peripheral blood mononuclear cells (PBMC) from each individual, obtained by ficoll-hypaque density gradient (Sigma, St. Louis, MO, USA). Viability was assessed by trypan blue exclusion (Sigma). Surface staining was done with antibodies against CD3 APC, CD4 PerCP and CD8 PE-Cy7 for 30 minutes at 4uC. After one wash, the cell membrane was permeated with Cytofix/Cytoperm solution for 20 minutes at 4uC, followed by washing with Perm/Wash 16 (BD Bioscience). Intracellular staining was done with anti-perforin FITC antibody (clone dG9) or mouse IgG2b isotype control (clone 27-35) for 30 min at 4uC. Lastly, cells were washed, re-suspended and analyzed as described above. At least 2610 5 CD3+ cells were recorded by flow cytometry and perforin expression was determined in CD4+/CD8+ gate.

Class I antigen specific double positive cells
As HLA-A2 is one of the most common class I allele in Colombia [23], donors were typed for HLA-A2 and subtype for HLA-A*0201 by flow cytometry and SS-PCR as previously described [24,25]. Tetramer analysis was only done in HLA-A*0201+ individuals, including: 13 chagasic donors (6 asymptomatic and 7 symptomatic) and 5 healthy controls. Cells were incubated with anti-CD3 APC, anti-CD4 PerCP, anti-CD8 PE-Cy7 and anti-HLA-A*0201 PE tetramers loaded either with T. cruzi or influenza virus derived epitopes. T. cruzi epitope or K1, is a 9 mer peptide (TLEEFSAKL) derived from N-terminal region (amino acids 4-11) of T. cruzi KMP-11 protein (Swiss-Prot accession number: Q9U6Z1) [24,25]. Influenza virus peptide was a modified epitope derived from the viral matrix proteins (a.a. 58-66) denominated MP-Flu (GILGFVTTL) [25]. Tetramers were synthesized by the National Institute of Health (NIH) Tetramer Facility (Atlanta, USA). At least 2610 5 cells were analyzed by flow cytometry in the CD3+ gate and tetramer expression was determined in the CD4+/CD8+ gates.
Cell stimulation, degranulation and IFN-c production PBMC from 5 asymptomatic (G0) and 6 symptomatic patients (two of each: G1, G2 and G3) were isolated using density gradient as described above. A total of 2610 6 PBMC were culture in the presence of anti-CD28 (1 mg/ml) and anti-CD49d (1 mg/ml) and one of the following conditions: medium alone, 10 mg/ml of K1 peptide or 3.7 mg/ml of Staphylococcal enterotoxin B (SEB). Cultures were incubated for 3 hours at 37uC with 5% of CO 2 , followed by an additional 6 hours of incubation in the presence of Brefeldin A 10 mg/ml (BD Pharmingen). For degranulation assays, anti-CD107a and CD107b FITC (clones H4A3 and H4B4, respectively) were added immediately following stimulation. For surface staining, antibodies for CD3-Pacific Blue (UCHT1), CD4-PeCy5.5 (SK3), CD8-APCH7 (SK1) and K1 PE tetramer were used. PBMC were washed once and then fixed/permeated using Cytofix/Cytoperm (BD Bioscience). Cells were washed twice with Perm/Wash 16 (BD Bioscience), stained with 10 ml anti-IFN-c PE-Cy7 (clone B27), rewashed once and re-suspended in FACs Flow (BD Bioscience). Population was gated according viability and CD3 expression. Expression of CD107 and production of IFN-c was based on the K1 tetramer positive CD4+/CD8+ cells. At least 1610 3 tetramer positive cells were acquired and analyzed in a FACS Aria with FACSDiva software (BD Bioscience).

Histology studies in cardiac tissue
Cardiac tissue from a chronic chagasic patient biopsy was used for immunochemistry. This specimen was obtained from a 47 year old male who was diagnosed with cardiac failure class III (NYHA classification) and underwent a cardiac transplant. Formalin-fixed and paraffin-embedded cardiac biopsy tissue was cut, deparaffinized in xylene and rehydrated with alcohol. Antigen retrieval was

Statistical analysis
Descriptive statistics (mean, standard deviation and percentages) was used to describe the populations and to present the flow cytometry data. Non-parametric analysis was carried out (Statistix 8.0 software) for groups comparisons by Kruskal Wallis (K-W) test followed by Dunn post hoc tests. Comparison of two groups was done by Mann-Whitney U test (M-W). Significance was considered a p value,0.05.

Phenotype analysis of double positive T cells
Cell frequency between chronic chagasic patients (CP) and healthy controls (HC) matched by age and gender were compared. The average percentage of total circulating DP T cells was higher (p = 0.0017) in CP (X = 2.1%60.9) compared with HC group (1.160.5) ( Figure 1B). Representative flow cytometry dot plots and gating strategy are shown in Figure 1A. To determine if this difference was attributed to the chagasic patients immune response and not to the cardiac damage or its pathological consequences, DP T cells percentages from CP were compared with those from donors with non-chagasic cardiomyopathy (NCC) (Figure 1A and 1B). Likewise, DP T cells frequency from CP was higher (p = 0.034) compared with NCC group (X = 1.260.4). For the following analysis, DP T cells were divided according to their phenotype into CD4 high /CD8 low and CD4 low /CD8 high . The frequency of CD4 high /CD8 low T cell was higher (p = 0.0058) in CP (X = 1.61%60.87) than in HC (0.8860.56) and NCC (0.8760.41) donors ( Figure 1C). Regarding to CD4 low /CD8 high T cell, significant differences were only observed between CP and HC (p = 0.015) frequencies ( Figure 1A and 1C).
To define the activation status of DP T cells in chronic Chagas patients, CD38 and HLA-DR surface markers were analyzed. Since, it was previously reported that CD4 high /CD8 low and CD4 low /CD8 high T cells differed in the expression of several surface markers, phenotypic analysis was done separately [1][2][3][4]10]. The percentage of DP T cells expressing HLA-DR was three times higher in CP in both CD4 high /CD8 low (p = 0.0031) and CD4 low / CD8 high (p = 0.01) populations than in the two control groups. No differences were observed between HC and NCC control groups ( Figure 2B). Representative flow cytometry density plots and percentages of HLA-DR positive DP T cells are shown in Figure 2A and Figure 2B, respectively. Analysis of CD38 in DP T cells showed that CD4 high /CD8 low subset had increased (p = 0.002) percentage of cell expressing this marker in CP (X = 38.9%622.0) compared with NCC (9.665.3), and not different with HC (20.6610.6). In contrast, the frequency of CD4 low /CD8 high expressing CD38 was higher in CP (X = 30.0%610.5) (p = 0.0001) when compared with both HC (12.667.7) and NCC (11.0610.5). When co-expression of both activation markers (CD38 and HLA-DR) was analyzed, CP doubled the percentage of activated cells compared to HC and NCC donors in both CD4 high /CD8 low (p = 0.009) and CD4 low /CD8 high T cells (p = 0.001) (Figure 2A and 2C). There were no differences when the analysis by clinical status was done. Due to the similarity of both subsets of DP T cells (Table S1), data is presented on the whole population in subsequent analyses.

Specificity and functional analysis of double positive T cells
To assess the cytotoxic potential of DP T cells, intracellular perforin was measured [10][11]. Increased (p = 0.002) percentages of cells containing perforin in CP (9.964.8) were found when compared with HC (3.363.2) and NCC (3.362.9) donors. Representative flow cytometry dot plots and percentage of perforin positive cells are shown in Figures 3A and 3B, respectively. No difference in perforin expression was found (p = 0.29) between asymptomatic (9.963.1) and symptomatic (9.965.7) chagasic patients.
Next, the antigen specificity of DP T cells was determined. To do so, the frequency of HLA-A*0201/peptide recognition was assessed using epitopes from T. cruzi and influenza virus, denominated K1 ( Figure 4A) and MP-Flu ( Figure 4B), respectively [25]. We found that both asymptomatic (X = 3.0%61.9) and symptomatic (4.963.6) CP had higher percentages (p = 0.0006) of circulating DP T cells specific for the K1 cytotoxic epitope than HC donors (0.660.4). There was no difference according to the disease stage (p = 0.267) ( Figure 4C and Table S2). The percentage of K1-specific DP T cells in the Chagasic donors was in average 27 times higher (p = 0.0005) than the mono-CD8+ K1-specific ones  Table S2. Representative flow cytometry dot plots and percentage of K1 specific DP T cells are shown in Figure 4A and Figure

Histology studies in cardiac tissue
Cardiac tissue from a chronic chagasic patient was analyzed in order to identify the presence of CD4+/CD8+ cells. The immunohistology analysis showed that myocardiocytes presented reparative nuclear changes such as bigger size, hyperchromatic and visible nucleoli. Moderate patchy myocardial infiltration mostly conformed by lymphocytes, some plasmocytes, macrophages and scarce eosinophils was also observed. The number of infiltrating lymphocytes was 10 or 12 per high power field, and they were mostly CD8+ cells (near 95%, Figure 6A and 6B) and some CD4+ cells ( Figure 6B). Double CD4+/CD8+ cells were found in the inflammatory infiltrate of the cardiac tissue in a frequency lower than 2%, as shown in Figure 6C.

Discussion
Circulating CD4+/CD8+ double positive (DP) T cells represent between 1-3% of the total T lymphocytes population [1]. However, previous evidence has suggested that this frequency can increase during several inflammatory diseases [2][3][4][5][6][7][8]. In this study we demonstrated that the percentage of peripheral DP T Double-Positive T Cells in Chagas Disease www.plosntds.org cells was higher in chronic chagasic patients compared with control donors, including individuals with non-chagasic cardiomyopathy. This finding differs from previous studies in HVC and HIV infected patients in which virus infected and control donors did not have differences in DP T cells frequencies on peripheral blood [10,26]. To the best of our knowledge, this research is the first report of an augmented percentage of DP T cell in human patients with a chronic parasitic infection.
Previous characterization of DP T cells indicates that this lymphocyte subpopulation is constituted mainly by terminally differentiated memory cells. A recent study showed that chronic chagasic patients had higher frequencies of CD4+ effectors memory T cells (T EM ) and CD8+ central memory T cells (T CM ) when compared with uninfected individuals [27]. Interestingly, in healthy donors CD4 high /CD8 low T cells were described as being T EM phenotype, meanwhile CD4 low /CD8 high were mainly T CM [10]. It will be interesting to determine if some of those CD4+ T EM and CD8+ T CM described in chagasic patients could include some DP T cells [27].
Similarly to DP T cells described in other human chronic infectious diseases with antigen persistence [10,26,28,29], we found that the expression of activation markers in these cells were increased in the chagasic donors. Consistently, some studies in T. cruzi infected donors have shown that their T cells (CD3+, CD3+/ CD4+ and CD3+/CD8+) tended to be more activated than cells from uninfected donors [30][31][32].
Regarding the source of the peripheral DP T cells, experimental data supports that they might either escape from the thymus [33] or represents over-stimulated mono-positive CD4+ or CD8+ T cells [34,35]. Interestingly, some studies have shown severe CD4+CD8+ thymocytes depletion coexisting with 16-fold increase of these cells in the periphery (subcutaneous lymph node) after T. cruzi acute infection in mice [21,36]. These findings support the ''thymus escape'' theory. However, expression of the ''second'' Double-Positive T Cells in Chagas Disease www.plosntds.org marker in mono-positive T lymphocytes as a consequence of antigenic over-stimulation seems also plausible in our study, given the consistent activation exhibited by the DP T cells [34]. More studies are needed to determine the origin of DP T cells in chagasic patients.
Perforin-mediated cytotoxicity has proved to be very important in the protection and pathogenesis of some parasitic infection, including cerebral malaria [37] and toxoplasmosis [38]. In animal models, there is evidence that implicates perforin expression with T. cruzi elimination during the acute disease [32,37]. However, perforin has also been involved in tissue damage during chronic Chagas disease [39][40][41]. After T. cruzi infection, perforinknockout mice had an increased parasite burden and increased number of IFN-c producing T cells infiltrating their hearts. However, these perforin-deficient animals showed more preserved cardiac tissue and less electric conduction abnormalities than normal littermates [39]. Even more, it has been suggested that human cardiac damage is directly related to an increase in the ratio of perforin-positive/total inflammatory cells in heart tissue [40].
Another remarkable result in this study is that DP T cells can recognized a T. cruzi derived class I epitope during chronic infection. The percentage of DP T cells specific for the K1 peptide was exceptionally high (1.7% to 11.5%) compared with previous reports of K1 specificity for mono-CD8+ T lymphocytes (0.09% to 0.34%) in a similar chronic chagasic population [25]. Likewise, in human viral infections (HCV and HIV) upon antigen exposure, DP T cells displayed a much higher frequency of cell-single cytokine production than CD8+ and CD4+ T cells [10,26]. Analysis of class I antigen recognition suggests that DP T cells in chagasic patients were parasite driven. Probably, as these peripheral cells are T. cruzi antigen-specific and have memory phenotype, they could migrate to the tissue where parasites persist and contribute to T. cruzi induced pathology. In fact, in situ cytotoxic lymphocytes (perforin or granzyme A positive cells) have been described in human heart [41] and gastrointestinal tract [42] of patients with T. cruzi induced tissue damage.
When activation markers, perforin and K1-recognition on DP T cells were compared in chagasic patients according to their clinical status (asymptomatic versus symptomatic patients), no differences were found. Nevertheless, it was notable that some symptomatic donors had higher percentages of K1 T. cruzi specific cells than asymptomatic ones, while percentages of recognitions for influenza virus epitope were similar. We also found that K1 specific DP T cells from symptomatic group displayed increased degranulation activity even with medium alone. However, very low percentages of these K1 tetramer specific DP T cells produced IFN-c after K1 peptide stimulation, as similarly described for CD8+ T cells in chronic chagasic patients [25]. This was not the case for the production of IFN-c after polyclonal stimulation, which was significantly augmented especially in the asymptomatic patients. Interestingly, K1 specific DP T cells that produce IFN-c did not display degranulating phenotype, indicating that DP T population is functionally heterogeneous and complex. In summary, K1 specific cells from asymptomatic donors had higher capacity of IFN-c secretion than cells from symptomatic donors which have greater cytotoxic potential.
In our study, we found a higher expression of perforin on peripheral DP T cells in the chagasic patients accompanied with an increased degranulation activity in the symptomatic ones. Also, it was demonstrated that DP T cells can migrated to the cardiac tissue. If the blood phenotype of these cells is maintained by the infiltrating ones, it might be possible to associate DP T cells with cardiac damage. A similar mechanism was suggested for HCV infected humans where DP T cells were found to infiltrate the liver [10].
Progressive loss of cytokines secretion is a characteristic of CD8+ T cells exhaustion, a phenomenon related to antigen persistence during chronic infections [43]. Indeed, IFN-c that is associated with protection against T. cruzi infection [44], is one of the last effector activities to be extinguished in this process [43]. So, our data suggest that K1 specific DP T cells, mainly from symptomatic donors, should be on the pathway of exhaustion, while they keep their cytotoxic potential. Lastly, as TNFa production has been associated with cardiac damage in Chagas disease [45], it will be of interest to test the production of TNFa on T. cruzi specific DP T cells.

Supporting Information
Table S1 Comparison of DP T cells among chagasic donors according to the presence of symptoms. Modified Figure 6. Staining for CD4 and CD8 in a chronic chagasic heart tissue. Immunochemistry for CD4 and CD8 staining in heart tissue from a chronic chagasic donor with cardiomyopathy. Slides were colored with hematoxyline/eosin to evaluate cellular infiltration and the presence of parasites. CD8+ cells yield a brown-color (arrows) and CD4+ a red-color end products (arrows head); DP T cells showed a red-brown color (wide arrow).