Table 3. BRSV-specific lymphocyte responses from tracheobronchial lymph nodes of ΔSHrBRSV vaccinated calves.

	FACSb
	CD4+IFNg+ (%)		CD8+IFNg+ (%)		Lymphocyteproliferation (COD)c	ELISAd
Calfgroupa	BRSVstim.	Controlstim.	BRSVstim.	Controlstim.		IFNγ (ng/ml)	IL-4 (ng/ml)
ΔSHrBRSV	0.27 (±0.13)*	0.16 (±0.07)*	0.09 (±0.04)	0.06 (±0.02)	0.14 (±0.12)*	1.10 (±1.08)*	0.24 (±0.28)
Control	0.22 (±0.10)	0.19 (±0.13)	0.05 (±0.03)	0.05 (±0.02)	0.02 (±0.03)*	0.20 (±0.10)*	0.32 (±0.27)

^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, OD_BRSV–OD_{cell 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.