Fig 1.
Poly I:C and Pam3CSK4 stimulate expression of surface receptors in B cells independent of T cell help.
CD19+ B cells were purified from spleens of C57BL6 mice and stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL), or the combination of both. Expression of surface receptor was determined after 24 hours by flow cytometry. (A) CD80 (n = 11), (B) CD86 (n = 7), (C) CD25 (n = 6), (D) MHC class II (n = 5), (E) CD69 (n = 4), (F) CD40 (n = 6). Data are shown as average ± SEM of individual B cell preparations as indicated, data is pooled from two to four independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.
Fig 2.
Poly I:C and Pam3CSK4 enhance T-cell-dependent B cell activation.
Purified B cells from C57BL/6 mice were stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both, with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. Expression of surface receptor was determined after 24 hours by flow cytometry. (A) CD80 (n = 8), (B) CD86 (n = 5), (C) CD25 (n = 6), (D) MHC class II (n = 7) and (E) CD69 (n = 3). Data are shown as average ± SEM of individual B cell preparations as indicated, data is pooled from two to four independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.
Fig 3.
Cytokine production enhanced by poly I:C and Pam3CSK4 stimulated B cells.
Purified B cells from C57BL/6 mice were stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both, with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. Supernatants were harvested after 24 hours and levels of (A) IL-6 (n = 6), (B) TNF-α (n = 6) and (C) CXCL10 (n = 8) determined by ELISA or CBA analysis. Data are shown as average ± SEM of individual B cell preparations as indicated, data is pooled from three to five independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.
Fig 4.
Proliferative response of B cells stimulated with poly I:C and Pam3CSK.
Purified B cells from C57BL/6 mice were stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both, with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. Proliferation was measured after 3 days by [3H]-TdR uptake. Data are shown as average ± SEM of 5 individual B cell preparations, data is pooled from at two independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.
Fig 5.
TLR3 and TLR2 Mediate B cell Response to poly I:C and Pam3CSK4 respectively.
B cells purified from TLR2-/- (A, C, E) or TLR3-/- (B, D, F) mice were stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL), the combination of poly I:C and Pam3CSK4, LPS (10 μg/mL) or CpG (25 μg/mL), with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. Relevant wild-type controls were tested in parallel (C57BL/6 for TLR2-/-, B6;129SF2/J for TLR3-/-). (A, B) Expression of CD80 was measured by flow cytometry after 24 hours. (C, D) Proliferation was measured by [3H]-TdR uptake after 3 days. (E, F) Production of TNF-α was measured by ELISA the supernatants harvested at 24 hours. Results are shown as the average ± SEM; TLR2-/- is from 4 individual B cell preparations pooled from two independent experiments; TLR3-/- is from 5 individual B cell preparations pooled from three independent experiments. Statistics compare the response of knockout B cells to corresponding wild type B cells and were calculated by 2-way ANOVA with Bonferroni post-test, *p<0.05, ***p<0.001.
Fig 6.
Allogeneic T cell response is increased in B cells stimulated with poly I:C and Pam3CSK4.
Purified B cells from C57BL6 mice and stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. After 24 hours, B cells were inactivated by mitomycin C, and resuspended at various concentrations. B cells were co-cultured with 100,000 CD4+ T cells isolated from BALB/c mice at ratios 1:10 (10,000 B cells), 1:25 (4,000 B cells) or 1:50 (2,000 B cells). (A) Proliferation was measured after 3 days by [3H]-TdR incorporation. Separate co-cultures were setup in parallel to detect IL-2 production (B) in supernatant as well as expression of CD25 (C) on CD4+ T cells after 3 days. Results are shown as the average ± SEM of 5 individual B cell preparations from 5 independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated.
Fig 7.
Antibody production and plasma cell marker expression are increased on B cells following T-cell-dependent stimulation with poly I:C and Pam3CSK4.
Purified B cells from C57BL/6 mice were stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. Surface expression of (A) CD138 (n = 7) and (B) TACI (n = 4) were determined by flow cytometry after 24 hours incubation. IgG was detected in supernatants harvested after 4 days of incubation by ELISA (n = 5). Results are shown as the average ± SEM of individual B cell preparations as indicated, data is pooled from two to three independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.
Fig 8.
Poly I:C and Pam3CSK4 adjuvant combination enhance antibody production by influenza and anthrax vaccines in vivo.
(A) CD-1 mice (n = 8) were vaccinated once with influenza recombinant hemagglutinin antigen (rHA; 0.5ug) formulated in DepoVax with no adjuvant, poly I:C (1 μg), Pam3CSK4 (1 μg) or the combination of both. (B) CD-1 mice (n = 8) were vaccinated once with anthrax recombinant protective antigen (rPA; 1 μg) formulated in DepoVax with no adjuvant, poly I:C (1 μg), Pam3CSK4 (1 μg), or the combination of both. For A and B, mice were bled on the indicated weeks after immunization and antigen-specific antibodies detected in serum by direct ELISA. Results are shown as endpoint titre ± SEM and are each representative of two independent experiments. Statistics by 2-way ANOVA with Bonferroni post-test comparing the combination to: “#” compared to no adjuvant, “+” compared to poly I:C; “*” compared to Pam3CSK4. No differences were detected between other groups.