Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Background:Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findings:We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/Significance:Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Atienza, Augusto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Perez Prados, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Buying, Alcinette. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Santos, Radleigh. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Tasso, Laura Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bonato, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Chiale, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pinilla, Clemencia. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Judkowski, Valeria A.. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning

Correction: Santos, R.S., et al. Improvement of IFNγ ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis. Cells 2015, 4, 1-18

The authors wish to make the following corrections to this paper [1]: [...

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning

Characteristics of patients with chronic Chagas' disease Cardiomyopathy.

a<p>F: female, M: male.</p>b<p>Patient's heart failure was classified according to New York Heart Association (NYHA) Functional Classification. Class I: Patients with cardiac disease with slight functional alterations but resulting in no limitation of ordinary physical activity; however, elevated activity causes symptoms, such as fatigue, palpitation, or dyspnea (shortness of breath); Class II: Patients with cardiac disease resulting in slight limitation of physical activity; comfortable at rest, but ordinary physical activity results in symptoms; Class III: Patients with cardiac disease resulting in marked limitation of physical activity; comfortable at rest, but less than ordinary activity causes symptoms; Class IV: Patients with cardiac disease resulting in marked limitation of physical activity, unable to carry out any physical activity without discomfort; symptoms of cardiac insufficiency at rest.</p><p>Patients with cardiac disease without any functional alteration were classified as Class 0.</p>c<p>The level of antibodies directed to <i>T. cruzi</i> lysate was determined by in-house ELISA as described upon <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#s2" target="_blank">Methods</a>.</p>d<p><i>T cruzi</i> lineage was analyzed by TSSA recognition as described upon <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#s2" target="_blank">Methods</a>. Results are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#pntd.0002906.s001" target="_blank">Figure S1</a>. ND: not detected.</p

Activation markers on CD4+ and CD8+ T cell subsets upon <i>T. cruzi</i> and ribosomal protein activation.

<p>PBMC isolated from patients with chronic Chagas' disease Cardiomyopathy (CCC; n = 27) and non-infected individuals (NI; n = 20) were seeded at 2.5×10⁶ cells/well and stimulated with <i>T. cruzi</i> lysate, P2β or CP0 proteins (10 µg/ml) or medium alone for 6 days. PBMC were stained with CD3-APC, CD4-PE-Cy5 or CD8-PE-Cy5 and activation marker-specific labeled antibodies (CD25-FITC and HLA-DR-PE) prior to flow cytometry analysis. 10,000–15,000 events in the lymphocyte gate (R1 gate) were acquired using a FACSAria flow cytometer (Becton Dickinson); dead cells were excluded by forward <i>vs</i> side-scatter (FSC/SSC) gating. A) Gate-pathway used to determine the activation expression in the populations graphed in B. B) Results were expressed as the percentage of CD25+ or HLA-DR+ cells in CD3+CD4+ (R2 gate) or CD3+CD8+ (R3 gate) lymphocytes. Horizontal lines represent the median and percentiles 25–75th, vertical lines represent percentiles 5–95th. Statistical analysis was performed using the Mann-Whitney U Test, ***<i>P<</i>0.001, **<i>P<</i>0.01, *<i>P<</i>0.05.</p

Parasite lysate but not ribosomal proteins trigger PBMC proliferative responses.

<p>PBMC isolated from chronic Chagas' disease Cardiomyopathy patients (CCC; n = 27) and non-infected individuals (NI; n = 20) were seeded at 2.5×10⁵ cells/well and stimulated with <i>T. cruzi</i> lysate, P2β or CP0 proteins (10 µg/ml) or medium alone for 6 days. Cell proliferation was determined by ³H-thymidine incorporation. Results are expressed as Stimulation index, calculated as: (mean cpm of stimulated cultures/mean cpm of non-stimulated cultures (medium only)). Each symbol represents data from a single subject. Statistical analysis was performed using the Mann-Whitney U Test, ***<i>P<</i>0.001.</p

Humoral response against ribosomal P proteins and their C-terminal peptides.

<p>The presence of antibodies directed against P2β and CP0 proteins as well as peptides R13, P015 and H13 in the sera of 27 patients with chronic Chagas' disease Cardiomyopathy patients (CCC) and 20 non-infected individuals (NI) was determined by ELISA as described under <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002906#s2" target="_blank">Methods</a>. Results are expressed as Reactivity index, calculated as: (Optical Density mean value obtained of each serum sample/baseline value). Each symbol represents data from a single subject. Statistical analysis was performed using the Mann-Whitney U Test, <i>***P<</i>0.001, *<i>P<</i>0.05. The line for each of the scatters represents the median.</p

Cytokine expression in PBMC from chagasic patients after in vitro stimulation.

<p>PBMC isolated from patients with chronic Chagas' disease Cardiomyopathy (CCC; n = 10) and non-infected individuals (NI; n = 8) were cultured in the presence of the indicated stimulus. Supernatants were collected on days 1, 2 and 6 and cytokines were quantified by multiplex technology. The Fold increase (FI) was calculated as: [(cytokine in stimulated culture) - (cytokine in NS culture)]/(cytokine in NS culture), where NS denotes non-stimulated cultured PBMC. The maximum FI out of the 3 day-determinations for each subject and for each cytokine are shown. In color are denoted the cytokines for which the FI in the CCC patients were statistically significantly higher than in non-infected individuals. Each symbol represents data from a single subject. Statistical analysis was performed using the Mann-Whitney U Test, ***<i>P<</i>0.001, **<i>P<</i>0.01, *<i>P<</i>0.05.</p