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

<div><p>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 <i>Trypanosoma cruzi</i> 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 <i>in vivo</i> 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 <i>in vitro</i> culture and stimulation conditions. Subsequently, cells were tested for their capacity to respond against <i>T</i>. <i>cruzi</i> lysate by measuring [³H]-thymidine incorporation and interferon-γ and GM-CSF secretion. Results allowed us to adjust initial <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> 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 <i>T</i>. <i>cruzi</i> specific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells.</p>