Enhancement of CD4⁺ T cell proliferation by Hsp70 complexes is antigen-specific and requires complex formation. (A) Monocytes isolated from FMD17 were co-incubated with autologous CD4⁺ T cells and either Hsp70 (1 μg/mL) alone, or Hsp70 complexed to FMDV 25-mer peptide BC2 (5 ng/mL) or a classical swine fever virus (CSFV) peptide (5 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) Monocytes isolated from FMD7 (A18/A19) which had previously been vaccinated against FMDV, but did not recognise the BC2 peptide, were co-incubated with autologous CD4⁺ T cells and antigens as indicated. Proliferation was assessed as above. (C) Monocytes isolated from FMD17 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 either pre-incubated to form a complex or added together without pre-incubation. Significant differences in proliferation between Hsp70:BC2 complex and Hsp70 + BC2 stimulated cells are indicated (*** p < 0.001). Proliferation was assessed as above.