INGEBI   02650
INSTITUTO DE INVESTIGACIONES EN INGENIERIA GENETICA Y BIOLOGIA MOLECULAR "DR. HECTOR N TORRES"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Generation of poly- and monoclonal memory T cell lines for antigen discovery in chronic Chagas disease
Autor/es:
ALCINETTE BUNYING; V JUDKOWSKI; NADIA SABRI; C PINILLA; ACEVEDO G; ATIENZA A; KA GOMEZ
Lugar:
Mar del Plata
Reunión:
Congreso; LXIV Reunion Anual de la Sociedad Argentina de Inmunologia (SAI); 2016
Institución organizadora:
Sociedad Argentina de Inmunología (SAI)
Resumen:
T cells are a major player in immune response against T. cruzi infection. Still, knowledge on the role of parasite-specific Th cells and their specificities is limited. Patient-derived antigen-specific T cell lines are a key tool for the study of this subject.The aim of this work was the generation of such lines from a chronic Chagas patient?s memory CD4 T cells by modulation of in vitro stimulation and culture conditions.CD3+ CD4+ CD45RO+ cells were magnetically sorted from PBMC and submitted to stimulation with T. cruzi lysate (antigen-specific) or PHA (non antigen-specific). At day 27 post-stimulation, cultures showing signs of proliferation were challenged with parasite lysate. Proliferation and Interferon (INF)-gamma secretion were used as a measurement of antigen-specific response. Autologous B-LCL were used as antigen presenting cells. Out of 192 wells initially stimulated with the lysate, 58.3% were steadily expanding at day 27, and most of these (87.5%) showed T. cruzi-specific response. Conversely, 100% of the PHA-stimulated wells reached this checkpoint, but only 37.5% responded to parasitic antigens. Nineteen T. cruzi-specific cultures were selected for further functional characterization by multiplex cytokine secretion assay (INF-gamma, TNF-alpha, GM-CSF, IL-2, -4, -10, -13, -17), and two were selected, on the basis of polyfunctionality, to undergo LDA. Seven hypothetically clonal, antigen-specific CD4 T cell lines were generated, which responded to T. cruzi lysate by INF-gamma and GM-CSF secretion. All of these, as well as the population from which they originated, maintained their specific response capability, even after long-term in vitro culture (225 days). TCR V-beta staining assay demonstrated that at least one of these lines is monoclonal, and expresses a member of the V-beta 5 family, which has a reportedly augmented frequency in chronic Chagas patients? CD4+ T cells.