Serum anti-LF IgG titers are significantly decreased following vaccination with LF + IL-1α in the absence of IL-1R1 on hematopoietic cells. Mice were vaccinated on days 0, +7, +21, and +42 after a 10-week rest period following bone marrow reconstitution. Serum samples taken on day +56 were tested by ELISA. Bars represent the geometric mean titers for each group on day +56 for all replicates, with error bars representing the 95% confidence level.*: CT groups do not differ significantly from each other and are significantly greater than LF alone (p < 0.001). 1: Significantly greater than LF +IL-1α within the same chimeric group (p < 0.001).2: Significantly greater than LF alone (p < 0.001). 3: Significantly greater than KO/KO LF + IL-1α (p < 0.001). 4: Significantly greater than KO/WT LF + IL-1α (p < 0.001). Four replicates unless noted; n = 7 for KO/KO LF alone (two replicates); n = 8 for KO/KO LF + CT (two replicates); n = 11 for KO/KO LF + IL-1α (two replicates); n = 18 for WT/WT LF alone; n = 19 for WT/KO LF alone, WT/WT LF + CT, and WT/WT LF + IL-1α; n = 20 for WT/KO LF + CT; n = 21 for WT/KO LF + IL-1α and KO/WT LF alone; n = 23 for KO/WT LF + CT; n = 24 for KO/WT LF + IL-1α.

LF + IL-1α-induced protection against lethal toxin is significantly impaired by the absence of IL-1R1 on hematopoietic cells. Mice were vaccinated on days 0, +7, +21, and +42 after a 10-week rest period following bone marrow reconstitution. Serum samples taken on day +56 were tested using an anthrax macrophage toxicity assay. Bars represent the geometric mean titers for each group on day +56 for all replicates, with error bars representing the 95% confidence level.*: CT groups do not differ significantly from each other and are significantly greater than LF alone (p < 0.001). 1: Significantly greater than LF +IL-1α within the same chimeric group (p < 0.05). 2: Significantly greater than LF alone within the same chimeric group (p < 0.001). 3: Significantly greater than KO/KO LF + IL-1α (p < 0.001). 4: Significantly greater than KO/WT LF + IL-1α (p < 0.01). Four replicates unless noted; n = 7 for KO/KO LF alone (two replicates); n = 8 for KO/KO LF + CT (two replicates); n = 11 for KO/KO LF + IL-1α (two replicates); n = 18 for WT/WT LF alone; n = 19 for WT/KO LF alone, WT/WT LF + CT, and WT/WT LF + IL-1α; n = 20 for WT/KO LF + CT; n = 21 for WT/KO LF + IL-1α and KO/WT LF alone; n = 23 for KO/WT LF + CT; n = 25 for KO/WT LF + IL-1α.

LF + IL-1α-induced antibody avidity is significantly decreased by the absence of IL-1R1 on hematopoietic cells. Mice were vaccinated on days 0, +7, +21, and +42 after a 10-week rest period following bone marrow reconstitution. Serum samples taken on day +56 were tested using an NH₄SCN elution ELISA. Lines represent titers as a percentage of the titer at 0 M NH₄SCN. *: Significantly less than WT/KO and WT/WT (p < 0.05). Four replicates. n = 10 for KO/WT LF alone; n = 13 for WT/KO LF alone; n = 14 for WT/WT LF alone; n = 19 for WT/WT LF + CT, WT/WT LF + IL-1α, KO/WT LF + CT, and KO/WT LF + IL-1α; n = 22 for WT/KO LF + IL-1α; n = 25 for WT/KO LF + CT.

Vaginal, not nasal, anti-LF IgA titers are significantly affected by the absence of IL-1R1 on hematopoietic and stromal cells. Mice were vaccinated on days 0, +7, +21, and +42 after a 10-week rest period following bone marrow reconstitution. Bars represent the mean IgA titer/μg on day +56 (vaginal) or day +66 or +82 (nasal lavage) with error bars representing the 95% confidence level. 1: Significantly greater than LF alone within the same chimeric group (p < 0.01).2: Significantly greater than KO/WT LF + IL-1α (p < 0.05).3: Significantly greater than KO/KO LF + IL-1α (p < 0.01).4: Significantly greater than LF + CT within the same chimeric group (p < 0.05).5: Significantly greater than WT/KO LF + IL-1α (p < 0.05). Four replicates unless noted. Fecal IgA: n = 7 for KO/KO LF alone (two replicates); n = 8 for KO/KO LF + CT (two replicates); n = 11 for KO/KO LF + IL-1α (two replicates); n = 15 for WT/WT LF alone; n = 16 for WT/WT LF + IL-1α; n = 18 for KO/WT LF alone and WT/WT LF + CT; n = 19 for WT/KO LF + IL-1α; n = 20 for WT/KO LF alone, KO/WT LF + CT, and WT/KO LF + CT; n = 24 for KO/WT LF + IL-1α. Vaginal IgA: n = 7 for KO/KO LF alone and KO/KO LF + CT (two replicates); n = 11 for KO/KO LF + IL-1α (two replicates); n = 16 for WT/WT LF + IL-1α; n = 17 for WT/WT LF alone; n = 18 for WT/WT LF + CT; n = 19 for WT/KO LF + IL-1α and LF + CT; n = 20 for WT/KO LF alone; n = 21 for KO/WT LF alone; n = 23 for KO/WT LF + CT; n = 25 for KO/WT LF + IL-1α. Nasal IgA (two replicates): n = 7 for KO/KO LF alone and WT/WT LF alone; n = 8 for KO/KO LF + CT and WT/WT LF + IL-1α; n = 9 for WT/WT LF + CT; n = 10 for WT/KO LF alone and LF + CT; n = 11 for KO/KO LF + IL-1α and WT/KO LF + IL-1α; n = 12 for KO/WT LF alone; n = 13 for KO/WT LF + CT and LF + IL-1α.

IL-1-responsive T and B cells are not required for IL-1α nasal adjuvant activity. Mice were vaccinated on days 0, +7, +21, and +63 after a 20-day rest period following adoptive transfer of splenocytes. Serum samples taken on day +77 were tested by ELISA. Bars represent the geometric mean titers for each group on day +77, with error bars representing the 95% confidence level. One replicate. N = 4 for KO/RAG LF alone and LF + CT; n = 5 for all other groups.

IL-1-responsive DCs are required for the maximal adjuvant activity of nasally delivered IL-1α. (A) Serum anti-rLFIgG titers on day +42 after intranasal immunization with rLF + IL-1α. (b) Serum anti-rLF IgG1 and IgG2c titers day +42 after intranasal immunization with rLF + IL-1α. Serum samples taken on day +42 were tested by ELISA. Bars represent the geometric mean titers for each group on day +42, with error bars representing the 95% confidence level. 1: Significantly greater than Il1r1^−/− and Myd88^−/− mice (p < 0.001).2: Significantly greater than all other groups (p < 0.05). 3: Significantly greater than Il1r1^−/− and Myd88^−/− mice (p < 0.01). One replicate. N = 18 for WT. N = 10 for all other groups.

Serum anti-rLFIgG subclass profiles are significantly altered following vaccination with LF + IL-1α in the absence of IL-1R1 expression on hematopoietic cells. Mice were vaccinated on days 0, +7, +21, and +42 after a 10-week rest period following bone marrow reconstitution. Serum samples taken on day +56 were tested by ELISA. Bars represent the geometric mean titers for each group on day +56 for all replicates, with error bars representing the 95% confidence level.*: CT groups do not differ significantly from each other and are significantly greater than LF alone (p < 0.001). 1: Significantly greater than LF + IL-1α within the same chimeric group (p < 0.01). 2: Significantly greater than LF alone within the same chimeric group (p < 0.05). 3: Significantly greater than KO/KO LF + IL-1α(p < 0.001). 4: Significantly greater than KO/WT LF +IL-1α(p < 0.01). Four replicates unless noted; IgG1, IgG2c, IgG2b, IgG3: n = 7 for KO/KO LF alone (two replicates); n = 8 for KO/KO LF + CT (two replicates); n = 11 for KO/KO LF + IL-1α(two replicates); n = 18 for WT/WT LF alone; n = 19 for WT/KO LF alone, WT/WT LF + CT, and WT/WT LF + IL-1α; n = 20 for WT/KO LF + CT. IgG1 and IgG2c: n = 21 for WT/KO LF + IL-1αand KO/WT LF alone; n = 23 for KO/WT LF + CT; n = 25 for KO/WT LF + IL-1α. IgG2b and IgG3: n = 20 for WT/KO LF + IL-1α; n = 21 for and KO/WT LF alone; n = 22 for KO/WT LF + CT. n = 25 for KO/WT LF + IL-1α(IgG2b) and n = 24 for KO/WT LF + IL-1α(IgG3).

Antigen-specific cytokine production after splenocyte restimulation with rLF is not impacted in IL-1R1 chimeric mice. Splenocytes were removed after the last sample collection on day +56 after four vaccinations. Bars represent the mean cytokine production for each group for all replicates minus the average production by unstimulated cells, with error bars representing the 95% confidence level. 1: Significantly greater than KO/KO LF + 4 μg IL-1α (p <0.05). 2: Significantly greater than KO/WT LF + 4 μg IL-1α (p <0.05). NS: Not significantly increased over LF alone within the same chimeric group. Four replicates unless noted; Four replicates unless noted; n = 5 for KO/KO LF alone, LF + CT, and LF + IL-1α (two replicates); n = 12 for WT/KO LF alone, WT/WT LF alone, and WT/WT LF + IL-1α; n = 14 for WT/WT LF + CT; n = 15 for WT/KO LF + CT and WT/KO LF + IL-1α; n = 17 for KO/WT LF alone and KO/WT LF + CT; n = 18 for KO/WT LF + IL-1α.

Mast cells are not required for the adjuvant activity of nasally delivered IL-1α. Serum anti-rLFIgG titers on day +35 after intranasal immunization with rLF with or without adjuvant. Mice were vaccinated on days 0, +7, and +21. Serum samples taken on day +35 were tested by ELISA. Bars represent the geometric mean titers for each group on day +35, with error bars representing the 95% confidence level. 1: Significantly greater than LF alone within the same mouse group (p < 0.001). One replicate. N = 5 for all groups.

IL-1-responsive DCs are required for the maximal induction of serum lethal toxin-neutralizing antibodies following vaccination with LF + IL-1α. Serum samples taken on day +42 were tested using an anthrax macrophage toxicity assay. Lines represent the geometric mean titer within each group, with circles representing the titer for individual mice within each group. 1: Significantly increased compared to all other groups (p < 0.01). One replicate. N = 18 for WT. N = 10 for all other groups.