Chagasic patients are able to respond against a viral antigen from influenza virus

In this work we assessed whether the CD8⁺ T cells from T. cruzi infected patients are able to respond to non-T. cruzi microbial antigens such as a well described epitope from influenza virus derived from the matrix protein. Specifically, it was studied whether the frequency, phenotype and functional capacity of Flu-specific CD8⁺ T cells are modified in chronic chagasic patients with respect to healthy donors used as control. The Flu-MP* peptide, which is an HLA-A*0201 restricted epitope was used as non-T cruzi epitope since HLA-A*0201 is the most prevalent human class I allele [27]. Consequently, subjects were HLA-A2 typed by flow cytometry and subtyped for HLA-A*0201 by PCR. It was found that 13 out of 50 chagasic patients (26%) were HLA-A2⁺; from whom all were HLA-A*0201⁺, classified as six chronic patients in indeterminate phase or A stage (normal electrocardiography ECG, and no major findings arising during clinical examination) and seven symptomatic chagasic patients having different disease severity levels as follows: 2 B (abnormal ECG results), 3 C (abnormal ECG results, decreased left ventricular ejection fraction, LVEF, and cardiac enlargement) and 2 D (abnormal ECG results, cardiac enlargement, decreased LEFV, and clinical signs of heart failure). Analysis of the twelve healthy donors showed that five of them (41.7%) were HLA-A2⁺, and HLA-A*0201⁺.

Tetramer consisting of HLA-A*0201/Flu-MP* peptide was used to evaluate the frequency of Flu-MP* specific CD8⁺ T cells in the thirteen HLA-A*0201⁺ chagasic patients and five HLA-A*0201⁺ healthy donors. Flu-MP* peptide specific cells were found in all chagasic patients, showing a range of frequency of 0.12% to 0.36% (mean 0.21%, SD = 0.07). Likewise, in the group of healthy donors all individuals had Flu-MP* peptide specific cells with a frequency range of 0.12% to 0.29% (mean 0.17%, SD = 0.07). There was no difference when all the groups of patients were compared based on their clinical status (indeterminate and cardiac chronic patients) or when chagasic patients were compared with healthy donors (P >0.05) (Figure 1).

Several studies showed the importance of memory CD8⁺ T cells in controlling viral infections in mice and human models [28]. Consequently, the phenotype of the Flu-MP* specific CD8⁺ T cells from HLA-A*0201⁺ infected patients and healthy donors was determined by analyzing the expression of CCR7, CD62L, CD27 and CD28. It was found that the Flu-MP* peptide-specific CD8⁺ T cells were predominately T_EM (CCR7- CD62L-) in both indeterminate and cardiac chronic chagasic patients without statistically differences (P = 0.206). Moreover, no differences were found when the expression in chagasic patients and healthy donors were compared (P = 0.055). However, significant differences were observed when the percentages of Flu-MP* peptide specific T_CM and T_EM cells from chagasic patients or healthy donors (P = 0.0001, and P = 0.008, respectively) were compared (Figure 2). As the CD8⁺ T cell differentiation degree, based on CD28, and CD27 expression, well correlates with their functional capacity [29], the Flu-specific CD8⁺ T cell stage of differentiation was compared among donors. It was found that Flu-MP* peptide-specific CD8⁺ T cells are equally distributed in the early and intermediate/late differentiation stages without showing any enrichment of a specific population. There were no statistically significant differences between chagasic patients and healthy donors (Figure 3).

To assess the functional characteristics of Flu-MP* specific CD8⁺ T cells, the IL-2 and IFNγ cytokine production and cytotoxic capacity, measured by the perforin secretion and CD107a/b surface expression, were evaluated in PBMC from ten chagasic patients and 5 healthy donors. For perforin analysis, it was observed that Flu-MP* peptide specific CD8⁺ T cells from T. cruzi-infected volunteers had similar percentage of expression, ranging from 17.56% to 77.78% (mean 41.4%, SD = 20.60%), than that from the healthy group of donors which ranged from 30.3% to 61.5% (mean 50.62%, SD = 12.05%). The surface expression of CD107a/b was detected in Flu-MP* peptide specific CD8⁺ T cells from both chagasic patients and healthy donors, with percentages ranging from 0.52 to 5.71% (mean 2.50%, SD = 2.05%) and 0.47% to 24.2% (mean 12.01%, SD = 8.57%), respectively. Furthermore, no differences in percentage, mean fluorescence intensity (MFI) or integrated mean fluorescence intensity (iMFI) in CD107a/b between chagasic patients and healthy donors were observed (Figure 4A, 4B). IL-2 was detected in eight out of ten infected patients with a range from 0.21% to 7.37% (mean 1.87%, SD = 2.6%) and in all studied healthy donors with a range from 0.56% to 1.57% (mean 1.09%, SD = 0.42%). IFNγ production was detected in Flu-MP* specific CD8⁺ T cells with a range from 0.51% to 3.42% (mean 1.61%, SD = 1.13%) in nine out of ten chagasic patients and in all healthy donors with a frequency range of 1.33% to 3.07% (mean 2.06%, SD = 0.74%). Consequently, no differences in cytokines production were observed in terms of percentage, MFI or iMFI when both groups were compared (Figure 4C, 4D).

All individuals enrolled in the study were volunteers who signed the informed consent form and were clinically evaluated at Fundación Abood Clínica Shaio and the Instituto Nacional de Salud (Bogotá, Colombia). Fifty Chagas disease patients with positive results in both immunofluorescence assays (IFI) and enzyme-linked immunosorbent assays (ELISA) were evaluated. Patients were classified according to the classification of the Incorporating American College of Cardiology and the American Heart Association Staging [37, 38], as follows: twenty-one chronic chagasic patients at indeterminate phase of the disease with normal ECG and no major findings arising during clinical examination (group A), and twenty nine cardiac chronic chagasic patients with abnormal ECG, having different disease severity levels (groups B, C and D). The group of healthy donors consisted of twelve T. cruzi seronegative individuals residing in non-endemic areas with normal ECG and no major clinical findings during clinical examination. Approximately a total of 20 mL of blood was obtained from all individuals by venipuncture in heparinized tubes to separate peripheral blood mononuclear cells (PBMCs), EDTA tubes for DNA extraction and tubes without anticoagulant for serological tests (Vacutainer; Beckton-Dickinson, San José, CA, USA). This study was approved by the Research and Ethics Committees from the Facultad de Ciencias, Pontificia Universidad Javeriana and Fundación Abood Clínica Shaio.

An HLA-A*0201 restricted peptide derived from the influenza virus matrix protein_58-66 with a modification in the seventh position was used (GILGFVTTL) and referred as Flu-MP* [19, 34, 39]. Peptide was synthesized using 9-fluorenylmethiloxycarbonyl (Fmoc) chemistry with a 0.64 substituted rink amide resin and Fmoc amino acids [40]. The peptides were cleaved by treatment with trifluoroacetic acid (TFA)/triisopropylsilan (TIS)/H2O (95/5/5) for 1 h, then precipitated with cold diethyl ether and purified by reverse phase high performance liquid chromatography (HPLC). The molar mass of the peptides was determined by MALDI-TOF mass spectrometry. The lyophilized peptide was reconstituted in dimethyl sulfoxide (DMSO) and stored at −20°C.

Individual HLA-A2 typing was performed by incubating 50 μL of peripheral blood with HLA-A2-specific antibody BB7⋅2-FITC (Becton Dickinson, Mountain View, California, USA) for 20 min at 4°C, followed by additional 15 min incubation. After adding red blood cells lysis buffer and 1X PBS washing [41], data were acquired and analyzed using a FACSCanto II flow cytometer and analyzed using FACSDiva (BD Biosciences) software.

A total of 50–150 ng genomic DNA was extracted from peripheral blood using a GFX genomic blood DNA purification kit (Amersham Biosciences, Piscataway, NJ, USA), and amplified using the 296 (5′-GTGGATAGAGCAGGAGGCT-3′), and 302 (5′-CCAAGAGCGCAGGTCCTCT-3′) primers. The PCR reaction was carried out according to Lasso et al. 2010 in a Stomacher 3500 Thermal Cycler, PTC 100 (MJ Research, Watertown, MA, USA) [34]. The expected 489 bp amplified product was separated on ethidium bromide stained 1.5% agarose gels [42].

HLA-A2 PE-labeled tetramer loaded with the Flu-MP* peptide, was synthesized by the National Institute of Health (NIH) Tetramer Facility (Atlanta, USA). Peripheral blood mononuclear cells (PBMCs) of all individuals were prepared using Ficoll-Hypaque density gradient (Sigma, St. Louis, MO), adjusted to 1 x 10⁶ cells per tube and stained with tetramer at 0,5 μg/mL, anti-CD3-PerCP and anti-CD8-FITC (BD Biosciences, San José, CA, USA) for 20 min in the dark at room temperature [34]. After washing with staining buffer (1% fetal bovine serum in PBS 1X), cells were resuspended with 500 μL of PBS 1X. Data were acquired in a FACSCanto II flow cytometer and analyzed using FACSDiva software (BD, Bioscience, San José, CA, USA). Frequencies of Flu-specific CD8⁺ T cells are the result of the frequency obtained minus the background of unlabeled cells with the tetramer.

PBMCs were adjusted to 2 x 10⁶ cells per tube and labeled with tetramers at 0.5 μg/mL, anti-CD3-PerCP, anti-CD8-APC-Cy7, anti-CCR7-PECy7, anti-CD62L-APC for 20 min in the dark at room temperature (all antibodies were purchased from BD Biosciences and BD Pharmingen, San Diego, CA, USA). After washing with 2 mL staining buffer (1% fetal bovine serum in PBS 1X), cells were fixed with 0.5% formaldehyde in PBS 1X. To determine intracellular perforin expression, after surface antigens staining, CD8⁺ T cells were permeabilized with Cytofix/Cytoperm (BD Pharmingen), washed twice with Perm/Wash^TM (BD Pharmingen) and stained with anti-perforin-FITC or mouse IgG2b κ isotype control for 30 min at 4°C. Intracellular cytokines were detected in PBMCs stimulated with Flu-MP* peptide in the presence of CD28 (1 μg/mL), and CD49d (1 μg/mL) for 12 h at 37°C. The last 9 h of culture were performed in the presence of brefeldin A (10 μg/mL) (BD Pharmingen). Then, cells were permeabilized and stained with anti-IFNγ-PECy7 and anti-IL-2-APC for 30 min at 4°C. To evaluate the cytotoxic activity, anti-CD107a and anti-CD107b-FITC were added to the PBMCs prior to stimulation. In each experiment, non-stimulated cells were used as negative control and Staphylococcal enterotoxin B (3.7 μg/mL) as positive control. Data were acquired in a FACSCanto II flow cytometer and analyzed using FACSDiva (BD Biosciences) software. Results were expressed as percentage, mean fluorescence intensity (MFI) and integrated mean fluorescence intensity (iMFI) obtained multiplying the frequency by the MFI. The latter was obtained multiplying the frequency by the MFI of tetramer positive CD8⁺ T cells [43].

Differences among groups were assessed by Mann Whitney test. GraphPad InStat 3.0 statistics software was used for analyses. P < 0.05 was considered statistically significant.