Evaluation of several flow cytometric assays for the analysis of T-cell responses in goats.
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.
D000276:Adjuvants, Immunologic; D000818:Animals; D004247:DNA / Q000096:biosynthesis; D004306:Dose-Response Relationship, Immunologic; D016995:Ehrlichia ruminantium / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006041:Goats / Q000276:immunology*; D008213:Lymphocyte Activation / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism; D014611:Vaccination; D014614:Vaccines, Synthetic
D000276:Adjuvants, Immunologic; D014614:Vaccines, Synthetic; D004247:DNA