Fig. 2. OmpX+LTA1 vaccine efficacy depends on CD4 T-cells and STAT3 expression by CD4 T cells.

(A) Vaccinated 6–10 week old male WT mice were intraperitoneally treated with 300 μg of isotype control or anti-GK1.5 Ab (to deplete CD4+ T cells) 2 days before immunization, which was administered weekly throughout experimental period to maintain CD4+ T cell depletion. Mice were challenged with 10⁴ CFU of the KP-396 strain, and lung and spleen CFU were assessed 24 h post challenge (n = 4–8, cumulative data of two separate experiments are shown). (B) To deplete lung resident CD4⁺ T cells, 500 μg of the GK1.5 Ab was intraperitoneally administered in combination with 100 μg delivered intratracheally at day 28 and 30 post-vaccination, followed by KP-396 challenge 24 h later. Lung and spleen CFU are shown (n = 7–9, cumulative data of two separate experiments are shown). (C) Lung and spleen CFU 24 h post infection in naïve WT C57BL/6 mice and OmpX+LTA1 immunized Stat3^fl/fl Cd4Cre⁻ mice and Stat3^fl/fl Cd4Cre⁺ mice (n = 7–9, cumulative data of two separate experiments are shown). (D) Representative dot plots of lung CD3⁺CD4⁺ T cells (left) or CD19⁺B220⁺ B cells (right), in OmpX+LTA1 immunized Stat3^fl/fl Cd4Cre⁻ mice and Stat3^fl/fl Cd4Cre⁺ mice. (E) Total lung CD4⁺ T cells and B cells in naïve WT C57BL/6 mice and OmpX+LTA1 vaccinated Stat3^fl/fl Cd4Cre⁻ mice and Stat3^fl/fl Cd4Cre⁺ mice (n = 4, representative from two independent experiments). (F) Frequency of OmpX-specific IL-17A-producing CD4⁺ T cells in naïve or immunized lungs from Stat3^fl/fl Cd4Cre⁻ mice and Stat3^fl/fl Cd4Cre⁺ mice was measured by ELISpot (n = 6, representative from 2 independent experiments). Data are presented as mean ± SEM. Significant differences are designated by using one-way ANOVA followed by Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.