Figure 1.
Proliferative response of mouse splenic B cells induced by TLR agonists in association with mCD40L.
Purified B cells were stimulated with TLR1/2 agonist (Pam3CSK4) (A), TLR2/6 agonist (Pam2CSK4) (B), TLR3 agonist (Poly (I:C)) (C), TLR4 agonist (LPS) (D), TLR7 agonist (R848) (E), TLR9 type C agonist (ODN 2395) (F) at the indicated concentrations and in association with increasing concentrations of mCD40L for 72 h. Proliferation was evaluated by measuring [3H]-thymidine uptake. Data are expressed as mean Stimulation Index (see material and methods) ± SD of three independent experiments. The mean of cpm in non activated B cells is 309±34. For C, D and F, although addition of mCD40L at all the concentrations induced significant changes, statistics are only shown for the concentrations used in further experiments (bold letters). *p<0.05, **p<0.01, ***p<0.001.
Figure 2.
B cell activation induced by TLR agonists in association with mCD40L.
Purified B cells were cultured with TLR1/2 agonist Pam3CSK4 (250 ng/mL), TLR2/6 agonist Pam2CSK4 (50 ng/mL), TLR3 agonist poly (I:C) (10 µg/mL), TLR4 agonist LPS (10 µg/mL), TLR7 agonist R848 (1 µg/mL) or TLR9 agonist ODN 2395 (200 nM) alone (light gray bars) or in the presence of 0.6 µg/mL mCD40L (dark gray bars). After 24 h, expression of activation markers CD69 (A) or CD86 (B) were measured by flow cytometry. Results are expressed as mean relative fluorescence intensity. Mean values ± SD of three independent experiments are shown. (C) CCL22/MDC levels were measured in cell culture supernatants by ELISA after 48 h. Results are expressed in pg/mL and represent means ± SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001.
Table 1.
Summary of B cell activation and/or differentiation induced by TLR agonist in association with mCD40L.
Figure 3.
Blimp-1 mRNA expression in splenic B cells induced by TLR agonists in association with mCD40L.
Purified B cells were cultured with the indicated TLR agonists (at the concentrations of figure 2) alone (light gray bars) or in the presence of 0.6 µg/mL of mCD40L (dark gray bar) (A) or with 10 µg/mL of TLR3 agonist poly (I:C), 10 µg/mL of TLR4 agonist LPS or 200 nM of TLR9 agonist ODN 2395 with or without 0.6 µg/mL of mCD40L and 1 µg/mL anti-IgM antibody, as indicated (B). After 72 h, total RNA was isolated and expression of Blimp-1 transcripts was evaluated by quantitative real-time PCR. Values were normalized to the mean of expression of three housekeeping genes: GAPDH, β-actin, HPRT. Results are expressed as the fold induction of gene transcription as compared to purified B cells cultured in medium. Data of one representative experiment out of three is shown. *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
B220 expression and AID mRNA expression in CD138-enriched purified B cell population after activation by TLR agonists in association with mCD40L.
Purified B cells were cultured with the indicated TLR agonists (at the concentrations of figure 2) alone (light gray bars) or in the presence of 0.6 µg/mL mCD40L (dark gray bar). After 72 h, the population was enriched for CD138 positive cells as described in Material and Methods. (A) CD138-enriched purified B cells were then co-stained with anti-B220 and anti-CD138 antibodies and the expression of these markers was determined by flow cytometry. Results are expressed as % of CD138+ B220low +/−SD of three independent experiments. (B) Total RNA was isolated and expression of AID transcripts was evaluated by quantitative real-time PCR. Results are expressed as the fold induction of gene transcription as compared to CD138-enriched purified B cells cultured in medium. *p<0.05, **p<0.01, ***p<0.001.
Figure 5.
Antibody production in purified B cell culture supernatant after activation by TLR agonists in association with mCD40L.
Purified B cells were cultured as described in figure 2 for 6 days. Cell culture supernatants were collected and amounts of IgM (A) and IgG (B) were measured by ELISA as described in Material and Methods. Results are expressed in µg/mL for IgM and ng/mL for IgG and shown as the means+SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001.