BACKGROUND: Flow cytometry (FCM) provides an alternative to radioactive methods for the analysis of T-cell responses. However, a comparative study of common FCM assays in an outbred ruminant model is lacking, which motivated this work. METHODS: Goats immunized with the obligate intracellular bacterium Cowdria ruminantium, inactivated and emulsified in oil-based adjuvants, were used as a model to study T-cell recall responses in vitro. FCM-based methods to measure Cowdria-induced lymphoblastogenesis, DNA synthesis, and interleukin-2 receptor (IL-2R) expression by T-cell subsets were compared. RESULTS: IL-2R expression was the most sensitive and reliable method provided that the number of molecules per cell was analyzed and not simply the percentage of positive cells of a given phenotype. Despite high background due to adjuvant and low proliferation, this method could detect antigen-specific activation of immune CD4(+) and CD8(+) T cells. CONCLUSIONS: FCM-based measurement of lymphoblastogenesis and DNA synthesis are not the most appropriate methods to analyze T-lymphocyte activation during vaccination of outbred animals. On several occasions, analysis of IL-2R expression was the only assay capable of discriminating between vaccinated and naive animals in this model. Copyright 2002 Wiley-Liss, Inc.
BACKGROUND: Flow cytometry (FCM) provides an alternative to radioactive methods for the analysis of T-cell responses. However, a comparative study of common FCM assays in an outbred ruminant model is lacking, which motivated this work. METHODS:Goats immunized with the obligate intracellular bacterium Cowdria ruminantium, inactivated and emulsified in oil-based adjuvants, were used as a model to study T-cell recall responses in vitro. FCM-based methods to measure Cowdria-induced lymphoblastogenesis, DNA synthesis, and interleukin-2 receptor (IL-2R) expression by T-cell subsets were compared. RESULTS: IL-2R expression was the most sensitive and reliable method provided that the number of molecules per cell was analyzed and not simply the percentage of positive cells of a given phenotype. Despite high background due to adjuvant and low proliferation, this method could detect antigen-specific activation of immune CD4(+) and CD8(+) T cells. CONCLUSIONS: FCM-based measurement of lymphoblastogenesis and DNA synthesis are not the most appropriate methods to analyze T-lymphocyte activation during vaccination of outbred animals. On several occasions, analysis of IL-2R expression was the only assay capable of discriminating between vaccinated and naive animals in this model. Copyright 2002 Wiley-Liss, Inc.
Authors: Alehegne W Yirsaw; Alexandra Gillespie; Fengqiu Zhang; Timothy P L Smith; Derek M Bickhart; Karthick P Gunasekaran; Maria Amir; Haeree Park; Janice C Telfer; Cynthia L Baldwin Journal: Immunogenetics Date: 2022-02-09 Impact factor: 2.846