Fig 4. Concentrations (pg/ml) of IFN-ɣ, IL-5, IL-10 and IL-17A in pools of culture supernatants of spleen cells obtained from mice (n = 5 per group) immunized with rM. vaccae.

Spleen cells obtained from immunized mice were cultured in triplicates in the absence of any stimulant (negative control) and in the presence of stimulants (experimental), i.e. pools of peptides (PPs) covering the sequences of individual immunizing proteins. The supernatants were collected on day 6. The culture supernatants from triplicates of negative controls and each stimulant were pooled separately and tested for secreted a) IFN-γ, b) IL-5, c) IL-10, and d) IL-17A cytokines in duplicate wells of 96-well plates by ELISA. The average values of cytokine concentrations from duplicates are presented in the figure. The concentrations of cytokines in response to various stimulants were considered significant with quantities >100 pg/ml and the ratio of Experimental/ Negative control (E/C)> 2 [31, 35]. Such values are marked with an asterisk (*).