Exploring immunological memory in Chagas disease: protocol optimization for the generation of Trypanosoma cruzi-specific human T-cell lines

ACEVEDO, GONZALO RAÚL; LONGHI, SILVIA; ATIENZA, AUGUSTO; CHIALE, PABLO; PINILLA, CLEMENCIA; JUDKOWSKI, VALERIA; GÓMEZ, KARINA ANDREA

Mar del Plata

Congreso; LXII Reunión Anual de la Sociedad Argentina de Inmunología; 2014

p { margin-bottom: 0.25cm; direction: ltr; line-height: 120%; text-align: left; widows: 2; orphans: 2; } Human Chagas disease, caused by infection with the protozoan Trypanosoma cruzi has an acute phase, followed by a chronic stage that can remain asymptomatic for years, or develop into digestive or cardiac pathologies, being the latter the most frequent form. Given the size and complexity of the parasite?s proteome and it?s interaction with the host?s immune system, positional scanning of combinatorial synthetic peptide libraries appears as a promising, high-throughput method for the study of immunological memory against T. cruzi. The aim of the present study was to produce an optimized protocol for the generation of pathogen-specific clonal T-cell lines, which peptide libraries are to be scanned with. For this, PBMC or CD3+CD8- sorted cells, from both chronic asymptomatic and cardiac patients, as well as from non-infected individuals, underwent different in vitro culture and stimulation protocols.Our results allowed us to adjust the following variables: a) for the cell culture and expansion stages, which intend to expand antigen-specific memory T-cell populations: initial stimulus concentration (2.5 μg/ml) and incubation time (1 day), initial culture density (250,000 PBMC/well), IL-2 addition in culture medium (since day 6, every 3-4 days), and b) for the challenge stages, by which cultures? response specificity for T. cruzi antigens was assessed: days since initial simulation (27 days), T-cells to antigen presenting cells ratio (1:2), T. cruzi lysate concentration (2.5 μg/ml) and readouts (Interferon-γ secretion, cell proliferation). When considered appropriate, flow cytometry experiments were carried out to find out whether protocol variations favored cytolytic or helper T-cell populations enrichment. These are the first steps in a novel, T-cell driven way towards the identification of potential prophylactic or therapeutic antigenic candidates, a matter of strategic relevance for the control of Chagas disease.