GLA and CpG combine to enhance T_H1 responses to ID93.

<p>Mice were immunized with ID93 adjuvanted with GLA, CpG, or GLA+CpG. (A and B) One week after the final immunization ID93-specific CD4⁺ T cells were identified by staining with I-A^b tetramers presenting dominant epitopes from Rv2608 and Rv3619. Cells are gated as singlet, CD4⁺ CD44⁺. Splenic ID93-specific T_H1 CD4⁺ T cells from immunized mice were identified by cytokine production following <i>ex-vivo</i> restimulation with ID93 and analyzed for (C) total cytokine response or (D) poly-functional responses. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083884#pone.0083884.s001" target="_blank">Figure S1</a> for representative cytokine staining. Data are representative of four experiments with similar results with 3–5 animals per group. ^*,^**,^***, and ^**** indicate <i>P</i><0.05, 0.01, 0.001, and 0.0001 respectively, relative to GLA+CpG as determined by ANOVA using the Bonferroni correction for multiple comparisons.</p

GLA and CpG combine to enhance the protective efficacy of ID93.

<p>Mice were immunized and challenged with a low dose of aerosolized <i>M.tb.</i> (A) four or (B) twelve weeks later. <i>M.tb.</i> burdens in the lungs were determined four weeks after infection. Statistically significant differences between vaccinated groups were determined by ANOVA using the Bonferroni correction for multiple comparisons. Data are representative of three experiments with similar results with seven animals per group.</p

Enhanced T_H1 responses to GLA and CpG are independent of TRIF signaling.

<p>(A) C57BL/6 and TRIF^−/− splenocytes were stimulated <i>ex vivo</i> with GLA, CpG or both and analyzed for IL-6, IL-12p40, and TNF production by macrophages. (B and C) C57BL/6 and TRIF^−/− mice were immunized with ID93 adjuvanted with GLA, CpG or GLA+CpG. Splenic ID93-specific T_H1 CD⁺4 T cells from immunized mice were identified by cytokine production following ex-vivo restimulation with ID93. Data are representative of two experiments with similar results with 3–5 animals per group. ^*,^**,^***, and ^**** indicate <i>P</i><0.05, 0.01, 0.001, and 0.0001 respectively, relative to GLA+CpG as determined by ANOVA using the Bonferroni correction for multiple comparisons.</p