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

IFNα is produced early after GLA-SE immunization.

<p>B6 mice were immunized with saline or ID93+GLA-SE. Sera and draining inguinal LNs were collected0, 6, 24, or 48 hours later and analyzed for IFNα protein expression by ELISA. Data are shown as mean ± SEM and are the combined results of two independent experiments with similar results with 3 or 4 mice/group. Statistics by unpaired t test; *p≤0.05 compared to Saline 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

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

IFNαR1 signaling contributes to T_H1 skewing.

<p>B6 mice were treated with IFNαR1 antibody or its isotype and immunized with ID93+GLA-SE and responses were analyzed one week after prime. (A) ID93 stimulated splenocytes were analyzed for the production of CD154, IFNγ, TNF and IL-5 by CD4 T cells. (B) One week after prime CD4 T cells were isolated and stained with an I-A^b tetramer presenting the dominant epitope for Rv3619 and analyzed for T-bet induction. (C) Sera were collected one week after prime and serially diluted to assess levels of anti-ID93 IgG1 and IgG2c. Data are shown as mean ± SEM of N = 5 animal/group and are from one experiment representative of two experiments performed except for IL-5 and T-bet levels measurement which were only done once. Statistics by simple or multiple t test corrected for multiple comparisons using the Holm-Sidak method between IgG or cytokine groups; *p≤0.05</p

IFNαR1 signaling is essential for lymphocyte activation and IFNγ production upon immunization with ID93+GLA-SE.

<p>B6 mice were treated with IFNαR1 antibody or its isotype and immunized with ID93+GLA-SE. Innate responses were assessed in the ipsilateral draining LN or in the contralateral LN by flow cytometry on LN cells or ELISA on LN supernatant The results are shown for the ipsilateral LN when not stated otherwise. (A) MFI and representative histograms of CD69 staining (B) IFNγ production assessed by ELISA, (C) IFNγR occupancy as indicated by decreased MFI of IFNγR staining with the monoclonal antibody GR-20 at 12 hrs after immunization. (D) IFNγ staining on CD8+ T cells. (E) IFNγ staining on NK cells. (F) IL-12 production assessed by ELISA. Data are shown as mean ± SEM of N = 4 animal/group and are from one experiment. Statistics by one-way ANOVA, *p≤0.05 compare to anti-IFNαR1 treated animals, #p≤0.05 compare to all the other groups.</p

CD94 is not necessary for clearance of LCMV, vaccinia virus or Listeria infection.

<p>(A) B6 and CD94-deficient mice were infected with 2×10⁵ pfu of LCMV (Armstrong strain). Spleens and livers were analyzed for LCMV titers on the indicated days. (B) B6 and CD94-deficient mice were challenged with 1×10⁵ cfu <i>Listeria monocytogenes</i>. Spleens and livers were analyzed for bacterial burdens three days later. (C) B6 and CD94-deficient mice were challenged with 5×10⁶ pfu vaccinia virus. Spleens and ovaries were analyzed for viral titers seven days later. Graphs represent the average ± s.e.m. of three to five animals per group per time point.</p

Splenic CD94-deficient and CD94^Tg/– NK cells are phenotypically normal.

<p>(A) Splenocytes from B6, CD94-deficient, CD94^Tg/–, and 129/SvJ mice were analyzed for CD94-NKG2 expression. CD94-NKG2⁺ cells were analyzed for NKp46 and TCRβ expression as shown in the second column. (B) NK cells (NKp46⁺ TCRβ^–) were analyzed for expression of CD94, NKG2A/C/E, NK1.1, Ly49C/I, and Ly49H. (C) CD19^– bone marrow cells were analyzed for NK cell precursors (NKG2D⁺ CD122⁺) and the developmental markers DX5, α_V, CD27, and CD11b. Data are representative of three to five experiments each.</p

CD94 is not necessary for control of MCMV.

<p>(A) Two days after infection with MCMV splenic NK cells from B6 and CD94-deficient mice were analyzed for expression of CD69 and intracellular IFN-γ and granzyme B. (B) CD94-deficient and CD94^Tg/– mice, (C) B6 mice treated with the non-depleting, blocking anti-NKG2A/C/E chimeric rat-mouse monoclonal antibody 20D5HCmIgG1-Q, anti-NK1.1 depleting antibody or PBS (D), or BALB/c mice receiving the 20D5HCmIgG1-Q antibody or PBS were infected with 5×10⁴ pfu MCMV and then analyzed for viral titers three days later. Graphs represent the average ± s.e.m. of four or five animals per group. Not statistically significant (n.s.).</p

CD94-deficient NK cells are educated and efficiently kill YAC-1 targets and MHC I-deficient splenocytes.

<p>(A) Splenocytes from B6, CD94-deficient, CD94^Tg/–, and 129/SvJ mice were assayed for degranulation as measured by surface staining for CD107a and intracellular IFN-γ production upon stimulated with plate-bound anti-NKp46 mAb. (B) Splenocytes from CD94-deficient mice and wildtype 129/SvJ mice primed with poly I:C <i>in vivo</i> were assayed for NK cell-mediated cytotoxicity against the YAC-1 target cell. (C) CD94-deficient mice and B6 mice either depleted of NK cells with anti-NK1.1 mAb or treated with PBS received a mixture of CFSE-labeled wildtype and <i>B2m^−/−</i> B6 splenocytes. Twenty-four hours later the frequency or CFSE-labeled donor cells in the spleen that were <i>B2m^−/−</i>was determined by expression of H-2K^b. Data are representative of three to five experiments each.</p