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. 2020 Oct 29;218(2):e20191393. doi: 10.1084/jem.20191393

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© 2020 Müller-Winkler et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 6. — BAFF depletion results in impaired recall responses due to loss of MBCs. (A) WT mice were immunized with CGG or NP-CGG in alum on day 0, followed by anti-BAFF or isotype control antibody injections 46 d and 50 d later and rechallenged with NP-CGG 16 d later. Blood was withdrawn 45, 66, 73, 80, and 87 d after primary immunization, and spleen and bone marrow were analyzed at day 87. (B) Mean (±SEM) numbers of NP⁺ IgM⁺ and IgG1⁺ MBCs in spleen and bone marrow and GC B cells in the spleen of mice immunized with CGG in alum (n = 8 for anti-BAFF and n = 12 for isotype control antibody in spleen and bone marrow) or NP-CGG in alum (n = 9 for anti-BAFF and n = 11 for isotype control antibody in spleen; n = 9 for anti-BAFF and n = 10 for isotype control antibody in bone marrow) as described in A, 21 d after rechallenge (gating as in Fig. S4 B). (C) Mean (±SEM) concentration of NP⁺ IgM and IgG1 in serum of mice immunized with CGG in alum (n = 8 for anti-BAFF and n = 12 for isotype control antibody) or NP-CGG in alum (n = 9 for anti-BAFF and n = 10 for isotype control antibody) treated as described in A. For clarity, the responses of mice primed with CGG or NP-CGG are shown separately as well as together. a.u., arbitrary unit. (D) Mean (±SEM) numbers of NP⁺ IgM⁺ and IgG1⁺ antibody-secreting cells (ASCs) in spleen (n = 3 for anti-BAFF and n = 6 for isotype control antibody for mice immunized with CGG in alum; n = 9 for anti-BAFF and n = 6 for isotype control antibody for mice immunized with NP-CGG in alum) and bone marrow (n = 3 for anti-BAFF and n = 7 for isotype control antibody for mice immunized with CGG in alum; n = 9 for anti-BAFF and n = 6 for isotype control antibody for mice immunized with NP-CGG in alum) of mice treated as described in A, 21 d after rechallenge with NP-CGG, determined by ELISPOT (Fig. S5 B). (E) Mixed bone marrow chimeras were generated by reconstituting irradiated Rag2^−/−Il2rg^−/− mice with 10% bone marrow from Baffr^+/+RCE or Baffr^fl/flRCE B6 mice (H-2K^b+) and 90% bone marrow from WT BALB/c mice (H-2K^d+) for 41 d. Mice were immunized with NP-CGG in alum on day 0, given five daily tamoxifen injections starting on day 35, and rechallenged with NP-CGG in PBS at day 58. Spleen and bone marrow were taken for analysis on day 79. (F) Mean (±SEM) numbers of H-2K^b+ or H-2K^d+ MBCs and PCs (n = 20 for Baffr^+/+RCE and n = 19 for Baffr^fl/flRCE; gating as in Fig. S5 D). Data pooled from two (B–D) and three (F) independent experiments. Mann-Whitney test was used for statistical analysis. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, 0.0001 < P < 0.001; ****, P < 0.0001.