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. 2024 Dec 30;15:10842. doi: 10.1038/s41467-024-55187-w

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 2 — C57BL/6 (WT) mice were i.p. injected with a single dose of AAI, AAII (7.5 mg/kg b.wt), or PBS (Ctrl). Mice were either immunized with NP-KLH in alum or left non-immunized (non-immun) 4 days post-AAI injection. At day 12 post-immunization, spleens were assessed for A NP-specific switched IgG1⁺ B cells (liveIgD⁻IgM⁻CD138⁻Gr1⁻B220⁺NP⁺IgG1⁺) by flow cytometry [Non-immun: Ctrl (3), AAI (3), AAII (3); NP-KLH: Ctrl (9), AAI (8), AAII (7)], B serum NP4 and NP27-specific IgG1 by ELISA [Non-immun: Ctrl (4), AAI (4), AAII (4); NP-KLH Ctrl (12), AAI (8), AAII (10)], and C NP4/NP27-specific IgG1 ratio was determined to measure antibody affinity maturation at day 12 post-immunization [NP-KLH: Ctrl (12), AAI (8), AAII (10)]. D NP7/NP27-specific IgG1 ratio was determined to measure antibody affinity maturation at day 21 post-immunization [NP-KLH: Ctrl (5), AAI (5), AAII (4)]. E NP4-specific antibody-secreting plasma cells generation in the spleen by ELISPOT assay [Non-immun: Ctrl (3), AAI (3), AAII (3); NP-KLH: Ctrl (7), AAI (7), AAII (6)]. F The frequency of TFh (liveCD4⁺CD44⁺CXCR5⁺PD1⁺) cells in the spleen was determined by flow cytometry at day 12 post-immunization [Non-immun: Ctrl (3), AAI (3), AAII (2); NP-KLH: Ctrl (8), AAI (7), AAII (6)]. AAN and control mice were primed with NP-KLH in alum followed by NP-KLH boost 36 days later. Primed only and boosted mice spleens were evaluated for G total GC (liveB220⁺GL7⁺CD95⁺) [Prime only: Ctrl (7), AAI (7), AAII (7); Prime-boost: Ctrl (10), AAI (14), AAII (8)], H NP-specific switched IgG1⁺ B cells (liveIgD⁻IgM⁻CD138⁻ Gr1⁻B220⁺NP⁺IgG1⁺) [Prime only: Ctrl (7), AAI (7), AAII (7); Prime-boost: Ctrl (9), AAI (12), AAII (7)], and I NP4-specific IgG1-producing plasma cells 6 days later [Prime only: Ctrl (7), AAI (7), AAII (7); Prime-boost: Ctrl (9), AAI (12), AAII (7)]. Each dot represents individual mice and data are pooled from at least 2–3 independent experiments (A–I). Data expressed as Mean ± SD. Statistical analyses by One-way ANOVA (A–I). A Ctrl vs. AAI ***P = 0.0007, AAI vs. AAII *P = 0.0252 and Ctrl vs. AAI ****P < 0.0001, AAI vs. AAII ****P < 0.0001. B Ctrl vs. AAI **P = 0.0069, AAI vs. AAII *P = 0.0247. C Ctrl vs. AAI ***P = 0.0004, AAI vs. AAII ***P = 0.0005. D Ctrl vs. AAI *P = 0.0565, AAI vs. AAII **P = 0.0063. E Ctrl vs. AAI *P = 0.020, AAI vs. AAII *P = 0.0299. F Ctrl vs. AAI ***P = 0.0007, AAI vs. AAII *P = 0.0276 and Ctrl vs. AAI **P = 0.0038. G Ctrl vs. AAI ****P < 0.0001, AAI vs. AAII ****P < 0.0001. H Ctrl vs. AAI **P = 0.0038, AAI vs. AAII **P = 0.0073. I Ctrl vs. AAI *P = 0.036, AAI vs. AAII *P = 0.0492. ns: statistically not significant. Source data are provided as a Source Data file.