Fig. 4. More efficacious induction of polyfunctional T cells by lentiviral vector compared to Ad5 vector.

C57BL/6 mice (n = 10/group) were immunized i.m. with 1 × 10⁷ IGU of Ad5-CMV-EsxH or 5 × 10⁷ TU of LV-β2m-EsxH, while negative control mice were injected with Ad5-CMV-GFP or LV-β2m-GFP. EsxH:3-11-specific CD8⁺ T cells were assessed at 28 or 90 dpi by cytometry. a Percentages of antigen-specific T cells within the CD3⁺ CD8⁺ T subset in the blood, as detected by PE-conjugated (K^b-IMYNYPAM)₄ tetramer (PE EsxH TET), at the indicated time points post-immunization. Shown are mean ± SEM. Smaller dots represent biological replicates. b Percentages of EsxH TET⁺ within the CD3⁺ CD8⁺ T splenocytes of the immunized mice at 28 or 90 dpi. Horizontal lines represent mean. Dots represent biological replicates. c Functional profile of EsxH:3-11-specific CD8⁺ T splenocytes from C57BL/6 mice immunized i.m with Ad5-CMV-EsxH or LV-β2m-EsxH (n = 5/group) at 28 dpi (top) or (n = 10/group) at 90 dpi (bottom), as investigated by ICS. The quality of response was characterized by the percentages of cells producing every possible combination of the measured cytokines (IFNγ, TNFα, and IL-2). Recapitulative frequencies of single, double, or triple positive CD8⁺ T cells at 28 or 90 dpi (left). The functional subsets were regrouped based on the number of effector cytokine produced (single (1+), double (2+), or triple (3+) positive cells (right)). Data are presented as mean ± SEM after subtraction of the background observed with the same splenocytes without peptide stimulation. Dots represent biological replicates. Statistical significance was determined via the Mann–Whitney t-test (**p < 0.01).