Figure 4

Antigen-specific and combination-specific Th1 cytokine and multifunctional T cell responses. Splenocytes were harvested at 14 days post last immunisation and re-stimulated for 6 h with A20 antigen presenting cells transfected with antigen-encoding plasmid DNA or incubated with synthetic peptide pools representing predicted CD8⁺ and CD4⁺ T cell epitopes for each antigen. Com1/4 = PY01533 + PY03832; Com2/3 = PY01606 + PY03673; Com1/2/3/4 = PY01533 + PY03832 + PY01606 + PY03673. Control = empty vector only or adjuvant only for DNA/protein or peptide/adjuvant groups respectively. Multi-parameter flow cytometry was used to quantify (A) total frequency of IFN-γ, IL-2 or TNF secreting CD4⁺ or CD8⁺ T cell populations analysed for each cytokine individually (i.e., total IFN-γ = cells producing only IFN-γ, or IFN-γ + IL-2, or IFN-γ + IL-2 + TNF; etc.) after (I) DNA/protein immunisation or (II) peptide/adjuvant immunisation. (B) Fraction of the total response represented by each of the eight possible combinations of IFN-γ, IL-2, or TNF cytokine secreting populations (triple positive, red; double positive, purple; single positive, orange; cytokine negative, grey). Data were analysed using FACS-DiVa software and the FlowJo population comparison tool and are presented as mean frequency (n = 5–15 mice pooled from up to 3 independent experiments) for each population after correction for background cytokine production in mock-stimulated cells. Error bars represent standard deviation (SD). Statistical comparison of immunised versus controls was performed using one-way ANOVA followed by Bonferroni’s posthoc test, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.