CD4+ T cell proliferation induced by monocytes to BC2 and Hsp70:BC2 complexes. (A) Monocytes isolated from cattle vaccinated against FMDV were co-incubated with autologous CD4+ T cells and FMDV 25-mer peptide BC2 at 0.5, 5 and 50 ng/mL. After 5 days, wells were pulsed with 37 kBq [3H] thymidine and incubated for a further 16 h before harvesting. Incorporated radioactivity was determined by liquid scintillation counting and expressed as counts per minute (cpm). Data are presented as the cpm × 103/min mean ± S.D. of triplicate cultures. (B–D) Monocytes isolated from cattle vaccinated against FMDV were co-incubated with autologous CD4+ T cells and either FMDV 25-mer peptide BC2 (5 ng/mL), Hsp70 (1 μg/mL) or BC2 and Hsp70, at the same final concentrations, but pre-incubated to form a complex. Responses to Pokeweed mitogen (PWM) and vaccine antigen are also indicated. Proliferation was assessed as above. One representative data set of three is shown for each of the three animals. Significant differences in proliferation between BC2 and Hsp70:BC2 stimulated cells are indicated (*** p < 0.001). (E) Monocytes were fixed with 0.5% paraformaldehyde before incubation with antigens and T cells. Significant differences in proliferation between Hsp70:BC2 stimulated cells using fixed or unfixed monocytes are indicated (*** p < 0.001). (F) 72 h after stimulation, supernatants from proliferation assays were assayed for IFN-γ production by capture ELISA. Data are presented as ng/mL mean ± S.D. of triplicate wells.