BCG vaccination induces polyfunctional CD4<sup>+</sup> but monofunctional CD8<sup>+</sup> T-cell response.

<p>(A–C) The lung and spleen cells of i.n. and s.c. BCG-vaccinated mice (<i>n</i> = 4/time point/group) were stimulated with WCL, and the magnitudes and polyfunctionality of CD3<sup>+</sup>CD4<sup>+</sup> and CD3<sup>+</sup>CD8<sup>+</sup> T cells in-terms of IFN-γ, IL-2 and TNF-α production were determined using polychromatic flow cytometry. (A) The longitudinal changes in the magnitudes of WCL-specific individual IFN-γ, IL-2 or TNF-α-producing cells among CD4<sup>+</sup> or CD8<sup>+</sup> T cells in the lung and spleen are plotted. (B) Representative dot plots show the frequency of WCL-specific IFN-γ and IL-2 or IFN-γ and TNF-α-producing cells among splenic CD4<sup>+</sup> and CD8<sup>+</sup> T cells from one mouse per vaccinated group at week 32 in comparison with age-matched naïve control, and the distributions of single, double and triple-cytokine-producers are illustrated. (C) The magnitudes of 7 possible combinations of WCL-specific cytokine-producing subsets constituting total cytokine<sup>+</sup> CD4<sup>+</sup> or CD8<sup>+</sup> T cells in the lung, spleen, cervical lymph node (CLN) and inguinal lymph node (ILN) are depicted. The data (A, C) are mean ± s.e.m. responses of 2 (at week 3, 78 and 104), 3 (at week 32) or 4 (at week 6 and 12) independent experiments.</p>