Fig. 2.
IgMlow anergic B cells paradoxically make more GC progeny. (A–C) CD45.1-marked HEL-specific B cells (105) that were anergic (An) or naïve (Na) were injected into normal C57BL/6 recipient mice together with 108 HEL-SRBCs or HEL2X-SRBCs. (A) Representative flow cytometric analysis of recipient spleen cells 5 d after immunization with HEL2X-SRBCs, gating on Fas+ CD38− GC B cells to enumerate percentage CD45.1+ donor-derived (Upper) or gating on CD45.1+ donor B cells (Lower) and measuring the percentage differentiated into B220low plasma cells with high intracellular HEL-binding antibody or into GC cells. (B) Arithmetic means and data from individual recipients immunized with HEL2X-SRBCs (exp 1–4) or HEL-SRBCs (exp 5 and 6). Statistical analysis was by t test: **P ≤ 0.01, ***P ≤ 0.001; n.s., not significant. (C) Representative immunofluorescence staining of a spleen cryosection from the recipient of anergic cells on day 5 after immunization with HEL2X-SRBCs, locating most HEL-binding progeny (red) in GCs. (D–G) Mature CD23+ B cells from C57BL/6 mice were stained with F(ab′) anti-IgM, and cells in the lowest (IgMlo; red histograms) or highest (IgMhi; blue histograms) quartile of cell-surface IgM were sorted and injected into B6.SJL-CD45.2 congenic recipients, and the recipients were immunized with SRBCs. (D) Distribution of surface IgM on the sorted populations compared with unsorted CD23+ B cells (gray-filled histogram) on day 0 (Upper) and compared with CD45.1+ recipient B220+ B cells 6 d after transfer (Lower). (E and F) Surface IgM mean fluorescence intensity (MFI) on the sorted populations from separate donors at the time of transfer (E) and in separate recipients after 6 d (F). (G) Frequency of CD45.2+ donor-derived cells among B220+ Fas− GL7− B cells (Left) or among B220+ Fas+ GL7+ GC cells (Center) in individual recipients, and the total number of donor-derived GC cells (Right). Statistical analysis was done as above.