Fig 2: MBCs do not extensively proliferate before differentiating to secondary PCs upon cognate antigen challenge. PNA−, B220+ MACS purified cells from Qβ immune (8 weeks post immunization) or naïve Ly5.1 mice were labeled with CFSE and transferred into congenic hosts. Recipient mice were challenged 24 h later with 50 μg Qβ VLPs i.v. (A) Representative FCM plot to identify Qβ specific CS B cells in the spleen 5 days after challenge. B220+ cells negative for IgM, IgD, CD4, CD8, CD1b, CD11c, and GR1 were analyzed for their Qβ binding. CFSE dilution was examined to prove proliferation of donor derived cells (Ly5.1+). (B,C) Number of Qβ specific CS B cells on day 4, 5, and 6 after challenge identified by FCM as B220+, negative for IgM, IgD, CD4, CD8, CD11b, CD11c, GR1, and binding Qβ. Qβ specific donor (B) derived cells were distinguished from host derived cells (C) using the Ly5 congenic marker. Mean with SEM. P values were obtained using a one-way ANOVA followed by Tukey's multiple comparisons test. **p < 0.01. n = 4 mice per group. Data representative of at least 2 independent experiments.

Fig 3: Adoptive transfer of Qβ VLP specific or naïve B cells and flow cytometric analysis of Qβ specific CS B and plasma cells in the spleen. (A) Congenic mice (Ly5.1 or IgHa) were immunized with 50 μg Qβ VLPs i.v. Eight weeks after immunization spleens of immunized and naïve mice were isolated and PNA− B220+ MACS purified cells were transferred into host mice (Ly5.2 or IgHb). Recipient mice were immunized with 50 μg Qβ VLPs i.v. 1 day after the transfer. Spleens, bone marrow, and serum were taken at several time points after challenge. (B) Representative FCM plots for the gating strategy to identify Qβ specific CS B cells in the spleen 5 days after immunization. B220+ cells not expressing IgM, IgD, CD4, CD8, CD11b, CD11c, or GR1 were analyzed for their binding of labeled Qβ VLPs. The congenic Ly5 marker was used to discriminate transfer from host derived CS B cells. (C) Representative FCM plots for the gating strategy to identify Qβ specific PCs in the spleen 5 days after immunization. B220low cells not expressing IgM, IgD, CD4, CD8, CD11b, CD11c, or GR1 were analyzed for their intracellular binding of labeled Qβ VLPs. The congenic Ly5 marker was used to discriminate transfer from host derived PCs cells.

Fig 4: Memory B cell derived secondary PCs produce antibodies of higher avidity. (A) MBC responses were initiated by vaccinating IgHa mice with 50 μg Qβ VLPs. After 8 weeks, PNA− and B220+ MACS purified B cells from memory (A) or naïve (B) donor mice were transferred into congenic recipients (IgHb). Recipient mice were challenged with 50 μg Qβ VLPs 24 h and 61 days after the transfer. The anti-Qβ IgG1 and IgG2a antibody titers in the serum were determined by ELISA on days 0, 2, 4, 6, 9, 12, 15, 21, 42, 61, 65, 68, and 85. Using Ha and Hb allotype specific detection antibodies, donor (IgHa), and host (IgHb) responses were discriminated. To determine the avidity index of IgGs in the sera after memory (C) or naïve (D) B cell transfer and VLP challenge, a modified ELISA was performed. Mean with SEM. P values were calculated using an unpaired t test. n = 4 mice per group. Data representative of 2 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Transfer of memory B cells leads to an increased number of PCs, which are rapidly induced but short-lived. Congenic Ly5.1 mice were immunized with Qβ VLPs to generate MBCs. Eight weeks after the immunization PNA−, B220+ MACS purified cells from memory or naïve mice were transferred into congenic hosts. One day after the transfer, recipient mice were challenged with Qβ VLPs and the anti-Qβ PC response in spleen and BM was elucidated by ELISPOT and FCM. Number of Qβ specific PCs in spleen (A) and BM (B) on day 4, 5, and 6 after challenge determined by ELISPOT. FCM analysis of Qβ specific PCs within the B220low, IgM, IgD, CD4, CD8, CD11b, CD11c, and GR1 negative compartment, by intracellular Qβ binding after membrane permeabilisation. Qβ specific donor derived PCs (C) were distinguished from host derived PCs (D) using the Ly5 congenic marker. Mean with SEM. P values were obtained using a one-way ANOVA followed by Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. n = 4 mice per group. Data representative of at least 1–2 independent experiments.