Methodological approach to the ex vivo expansion and detection of T. cruzi-specific T cells from chronic Chagas disease patients

The discovery of T cell epitopes is essential not only for gaining knowledge about host response to infectious disease but also for the development of immune-intervention strategies. In Chagas disease, given the size and complexity of the Trypanosoma cruzi proteome and its interaction with the host’s immune system, the fine specificity of T cells has not been extensively studied yet, and this is particularly true for the CD4+ T cell compartment. The aim of the present work was to optimize a protocol for the generation of parasite-specific memory T cell lines, representative of their in vivo precursor populations and capable of responding to parasite antigens after long-term culture. Accordingly, peripheral blood mononuclear cells (PBMC) from both chronic asymptomatic and cardiac patients, and from non-infected individuals, underwent different in vitro culture and stimulation conditions. Subsequently, cells were tested for their capacity to respond against T. cruzi lysate by measuring [3H]-thymidine incorporation and interferon-γ and GM-CSF secretion. Results allowed us to adjust initial T. cruzi lysate incubation time as well as the number of expansions with phytohemagglutinin (PHA) and irradiated allogeneic PBMC prior to specificity evaluation. Moreover, our data demonstrated that parasite specific T cells displayed a clear and strong activation by using T. cruzi lysate pulsed, Epstein-Barr virus (EBV)-transformed human B lymphocytes (B-LCL), as autologous antigen presenting cells. Under these culture conditions, we generated a clone from an asymptomatic patient’s memory CD4+ T cells which responded against epimastigote and trypomastigote protein lysate. Our results describe a culture method for isolating T. cruzispecific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells

Medical students maintain their humanistic and patient‑centred vocation throughout Medicine Degree in Spain: a study based on narratives

Narrative medicine has great educational potential in the degree of medicine. This study explores for the frst time the use of narrative medicine in relation to longitudinal evolution of medical vocation for the same group of students. In the context of the Degree in Medicine at the Universidad Autónoma de Madrid (Spain), students wrote narratives about what it meant to them to be a doctor at the beginning and end of their studies. The narratives of 338 students of the academic years 2012/13– 2017/18 and 2013/14–2018/19 were analysed and compared. Students mostly pursued a degree in medicine on account of humanistic motivations, which are reinforced throughout their degree. In contrast, up to 10% of students reference to have experienced vocational crises and sufered frustration, with up to 25% of the references pertaining to having made signifcant sacrifces. Students maintain and evolve their humanistic, patient-centred vision throughout their degree studies, despite the difculties they appear to encounter. We suggest that eforts must be made to include more humanistic perspectives in the medical degree to keep this trend, which may improve both the educational experience created in universities and the health care given to patient

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

Correction: Methodological approach to the ex vivo expansion and detection of T. cruzi-specific T cells from chronic Chagas disease patients.

[This corrects the article DOI: 10.1371/journal.pone.0178380.]

Effect of stimulus used for initial expansion of antigen specific memory CD4⁺ T cells and their <i>in vitro</i> response against <i>T</i>. <i>cruzi</i> antigens.

<p>Sorted memory CD4⁺ T cells from an asymptomatic Chagas patient (RM30) and a non-infected subject (MM) were stimulated with PHA (B, D) or parasite lysate (C, E) as detailed under Materials and Methods. Five thousand cells from each culture well were challenged between days 27–32 (depending on cell growth) with parasite lysate or culture media only and the antigen-specific response was measured as IFN-γ secretion and proliferation. Ten thousand autologous overnight-primed B-LCL per well were used as APC. SI was calculated for each well as the response against <i>T</i>. <i>cruzi</i> lysate divided by the cells baseline response (media only condition). Statistical analysis of the percentage of positive wells is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178380#pone.0178380.s006" target="_blank">S3 Table</a>. <b>A</b>. Timeline representation of stimulation and challenge protocols. Numbers in brackets next to symbols indicate the day (or range of days) since protocol start at which each step was performed. <b>B, C</b>. Pie chart representations of the wells that showed cell growth after initial stimulation, and different degrees of antigen specific response against <i>T</i>. <i>cruzi</i> lysate. <b>D, E</b>. SI values (scatter plots) and percentage of positive wells (bars) for each readout. Cultures considered positive were those with an SI≥2 (dotted line). Positive and total studied wells are indicated in numbers above the bars.</p

Antigen specific response in selected cultures from subject RM30.

<p>Cell lines were established from <i>T</i>. <i>cruzi</i> specific cultures, based on results from experiment depicted on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178380#pone.0178380.g003" target="_blank">Fig 3</a>. Cells were submitted to 2 PHA expansion cycles prior to specificity evaluation. Five thousand cells from each culture well were challenged with <i>T</i>. <i>cruzi</i> lysate, using 10⁴ autologous overnight-primed B-LCL per well as APC. SI was calculated for each well as the response against <i>T</i>. <i>cruzi</i> lysate divided by the cells baseline response (media only condition), cultures considered positive were those with an SI≥2 (dotted line). Bars show the mean values and standard deviation for three replicates of each measure.</p

Antigen specific response in cultures resulting from limiting dilution assay (LDA) of culture RM30.II.

<p><b>A</b>. Twenty-five thousand cells from each culture well were challenged with <i>T</i>. <i>cruzi</i> lysate, using 5×10⁴ autologous overnight-primed B-LCL per well as APC. Specificity against <i>T</i>. <i>cruzi</i> antigens was assessed for several potentially monoclonal lines by IFN-γ and GM-CSF secretion. N/A: Non applicable. <b>B</b>. Specific response was tested using lysates from different stages in the parasite’s life cycle (epimastigote and trypomastigote/amastigote) for T cell lines RM30.II.84 and .85.</p

Detected frequencies of TCR-Vβ families expressed on T cells from lines RM30.II and RM30.II.84.

<p>Detected frequencies of TCR-Vβ families expressed on T cells from lines RM30.II and RM30.II.84.</p