BRSV-specific lymphocyte responses from tracheobronchial lymph nodes of ΔSHrBRSV vaccinated calves.

aFour groups of 5 calves were vaccinated as described in Fig. 1 and challenged with BRSV, 5 weeks after vaccination, on post-infection day (PID) 0. Lymphocytes were isolated on PID 7 from tracheobronchial lymph nodes of calves vaccinated with ΔSHrBRSV and controls, and stimulated ex-vivo with either BRSV-infected or uninfected cell lysate (heat-inactivated).bAfter 18 hours of incubation, production of IFNγ by CD4+ and CD8+ lymphocytes were assayed using a flow cytometer (FACSVerse, BD Biosciences) and data were analyzed using FACSuite software (BD Biosciences). Results are expressed as % of CD4+ or CD8+ cells producing IFNγ.cAfter 7 days of incubation, proliferative responses were determined by corrected optical density (COD) of Alamar Blue (Invitrogen, Sweden). Results are expressed as the mean corrected OD (COD, ODBRSV–ODcell lysate).dAfter 9 days of incubation, IFNγ and IL-4 were analysed in supernatants of BRSV-restimulated cells by ELISA (BioRad).*Statistically significant difference between groups is indicated by asterisks; p≤0.05 (*). Standard deviations are presented within parenthesis.