Fig. 5. Effects on TFH and GC B-cell responses in neonatal mice following oral priming immunization with CTA1-DD adjuvant.

Neonatal mice were immunized on day 5 by feeding 5 µg NP-OVA with or without 10 µg CTA1-DD and boosted 30 days later with 5 µg NP-OVA administered via oral gavage. The induction of GC B cells and TFH cells was assessed in the PPs at 10 days after priming (a, c) or in mesenteric lymph nodes (MLN), PPs, or the sub-mandibular lymph node (smLN) at 5 days after an NP-OVA booster dose. b, d–h Flowcytometric analysis of the frequency and absolute number of GC B cells after priming (a) and after a booster immunization (b) and TFH cells after priming (c) and after a booster immunization (d). e Flowcytometric analysis of the percentage of GC B cells in the PP expressing Ephrin-B1 (EBI) or CD73 and f the frequency and number of plasma cells (CD138⁺) in the PP induced by CTA1-DD adjuvant. g Enhanced high-affinity NP-specific antibodies are found in serum from CTA1-DD adjuvanted immune responses. The mean ratio ± SD is given for log₁₀ titers of high-affinity (NP4) over low-affinity (NP30) IgG antibodies. h Flowcytometric analysis of the percentage of IgA⁺ GC B cells in the MLN and PP or CD138⁺ plasma cells in the PP following CTA1-DD adjuvant (black bar and + ) or in unadjuvanted responses (gray bar and –). i Lack of adjuvant effect of CTA1-DD in adult mice following oral immunization. OVA-specific intestinal IgA, serum IgA or IgG, and OVA- or CT-specific spot forming cells (SFC) in the lamina propria (LP) after 10 days following p.o. immunizations (3 × ) of adult mice with 10 mg OVA in the presence of cholera toxin (CT) or CTA1-DD adjuvant. All graphs are based on pooled data from 2–3 independent experiments with 2–5 mice in each group and results are given as means ± SD. A statistical analysis was performed using two-way ANOVA followed by Dunnet correction for multiple comparison; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.