Figure 3.

Polyfunctional CD4⁺ and CD8⁺ T cells in (A) immune cells isolated from lung (B) splenocytes of BCG-, rBCG-Mkan85B-, or rBCG-Mkan85B/DNA-Mkan85B-vaccinated CB6F1 mice. The induction of polyfunctional CD4⁺ T cells by stimulation with peptide 25 (left panel) and the induction of polyfunctional CD8⁺ T cells by stimulation with Pep8 (right panel) are shown. The data represent the results from three independent experiments with two to four mice per group. * p < 0.05. ** p < 0.01. (C) The gating strategy used to identify IFN-g-, IL-2- and TNF-producing CD4⁺ and CD8⁺ T cells among immune cells isolated from lung and splenocytes from a representative mouse is shown. The upper 4 panels show the initial gating of total events, including a singlet cell gate, followed by selection for lymphocytes. Live CD3⁺ T cells were identified by negative LIVE/DEAD staining and CD3 expression. CD4⁺ and CD8⁺ T cells were further identified by CD4 or CD8 expression. Antigen-specific IFN-g-, IL-2- and TNF-producing CD4⁺ and CD8⁺ T cells were gated as shown. The cells producing three, any two and only one cytokine were determined by Boolean combinations. Each cytokine-positive cell was assigned to one of seven possible combinations of these three cytokines (D), and the total number of cytokine-producing cells was calculated as the percent of CD4- and CD8-positive cells. The sum of the three- and two-cytokine-producing cells was measured as polyfunctional T cells specific for epitope peptides (D).