FIG. 4.

(A) Lymphocyte proliferation assay. Splenocytes were prepared 1 month after the last immunization and were cultured with cOTC (final concentration, 1 or 10 μg/ml) for 72 h. Splenocyte proliferation was observed by adding Alamar Blue for 15 to 18 h and was calculated as described in Materials and Methods. Results are expressed as mean specific absorbance (at 570 to 600 nm) and represent means ± standard errors. Significant differences between the antigen group (cOTC plus Freund's adjuvant) and the PBS group, determined by the Student t test, are indicated by asterisks (P < 0.05). (B) IFN-γ and IL-2 production in cultured spleen cells of mice injected with cOTC plus Freund's adjuvant or the PBS control. Results are expressed in picograms per milliliter and are means ± standard errors. Significant differences obtained by comparison with unstimulated cells, as determined by a paired Student t test, are indicated by asterisks (P < 0.05).