Fig 1.
ID93+GLA-SE TH1 response is dependent on T-bet and IL-12 whereas antibody production is only dependent on T-bet.
B6, IL12-/- and T-bet-/- mice were immunized with ID93+GLA-SE. (A)One week after boost, splenocytes were isolated and stimulated with ID93. CD4 T cells were analyzed for the production of CD154, IFN-γ, and TNF. (B) Sera were collected three weeks after the first immunization and serially diluted to assess levels of anti-ID93 IgG1, IgG2c and IgG Total. Data are shown as mean ± SEM of N = 4–5 animal/group and are from one experiment representative of two experiments performed. Statistics by two-way ANOVA with Dunnett’s correction for multiple comparison test within IgG and cytokine groups versus B6; *p≤0.05.
Fig 2.
IFNα is produced early after GLA-SE immunization.
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.
Fig 3.
IFNαR1 signaling is essential for lymphocyte activation and IFNγ production upon immunization with ID93+GLA-SE.
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.
Fig 4.
IFNαR1 signaling contributes to TH1 skewing.
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-Ab 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