Fig 2: GC function is required for cross-strain protective humoral responses.a Top panel indicates the design of the BALF transfer experiment for protective antibody in Bcl6-deficient mice. Mice intranasally administered with BALF or serum (60 µg/ml anti-Narita IgG) from WT (white), Bcl6ΔT (black) or Bcl6ΔB (red) mice infected with A/Narita/1/2009 were challenged intranasally with a lethal dose of A/Narita/1/2009 (left) (BALF WT n = 3, BALF Bcl6ΔT n = 4, Bcl6ΔB n = 4, Serum WT n = 3, Bcl6ΔT n = 5, Bcl6ΔB n = 6) or A/PR/8/1934 (right) (WT n = 8, Bcl6ΔT n = 10, Bcl6ΔB n = 4, Serum WT n = 4, Bcl6ΔT n = 5, Bcl6ΔB n = 6). The data indicate body weight change. Missing symbols mean that the mice were euthanized, as described in Fig. 1b. b A/H1N1pdm09-HA- and A/PR/8/1934HA-specific IgG1 and IgG2 titers in BALF from WT (white bars), Bcl6ΔT (black bars), and Bcl6ΔB (red bars) mice (n = 7 each) at 14 days post-infection with A/Narita/1/2009 were measured by ELISA. c The data indicate single or dual-binding B cells in the B220+IgM−IgD−Dump− population from MLN of WT (white bars, n = 4), Bcl6ΔT (black bars, n = 5), and Bcl6ΔB (red bars, n = 7) mice. UI represents unimmunized. Bars represent mean; *,#p < 0.05, **,##p < 0.01 by unpaired, two-tailed t-test (WT group with other groups respectively). All error bars represent SEM.

Fig 3: Reduced BCR Levels on DZ GC B Cells(A and B) Mice were immunized with SRBCs and splenic IgDlow CD95+ GL7+ GC B cells were analyzed on day 7. (A) BCR levels on DZ populations (CXCR4high) were analyzed and compared to B220neg non-B cells. The gate indicates a BCRlow DZ population. (B) DZ and LZ cells are gated as shown and BCR levels overlaid with IgD+ CD95low follicular B cells.(C–G) HEL-specific SWHEL or MD4 B cells were adoptively co-transferred with OT-II CD4+ T cells and mice were immunized with HEL-OVA/adjuvant adjuvant. Splenic IgDlow CD95+ GL7+ CD45.1+ GC B cells were analyzed on day 7. (C) Surface and intracellular BCR (IgMa) levels were co-stained in fixed/permeabilized MD4 GC B cells revealing a subset of cells that is negative for both. (D and E) DZ cells with low BCR levels in MD4 transgenic (D) and SWHEL gene targeted GC B cell populations (E). (F and G) Single CXCR4high BCRhigh and CXCR4high BCRlow DZ SWHEL GC B cells (gated IgDlow, CD38low, GL7+, CD45.1+, or CD45.2+) were index sorted and cultured for 8 days on NB21 feeder cells. (F) The BCR and CXCR4 staining profiles (at the time of sort) of single GC B cells that grew and secreted detectable antibody after culture are shown. The plots for cells that did not grow, or that grew did not secrete detected antibody, are also shown and overlaid with the total GC B cell population for reference. Plots for additional mice are shown in Figure S1. (G) Summary of results. Numbers of cells in each group per experiment are indicated.(H) Surface BCR levels on Aicda+/+ and Aicda-/- IgDlow CD95+ GL7+ GC cells from mixed bone marrow chimeric mice 8 days after SRBC immunisation. Numbers in (C–E) and (H) represent mean ± SEM from 7 (C and D), 10 (E) and 11 (H) mice from 2–4 experiments. See also Figure S1.

Fig 4: Memory B cells expand in and around pre-existing germinal centres on reactivation, but most cells do not acquire GL7 expression.(a) One year after oral priming immunizations, the proportion of NP-specific memory B cells in various tissues was monitored kinetically following a single p.o. challenge immunization with NP-CT. The frequency of NP-specific GFP+ cells among total CD19+B220+CD138− B cells at given time points was determined in individual mice (x; protocol as in Fig. 3a). (b,c) Flow cytometry analysis of GL7 and IgD expression on GFP+ memory B cells following the p.o. challenge. (b,c) The proportion of GFP+ cells that were GL7+ IgD− was determined using the indicated gates. (c) The percentage of GFP+ memory B cells that were GL7+IgD− was low in the PP, while it was two- to threefold higher in the MLN. (d,e) Antibody-labelled frozen sections of the PP and MLN were prepared 7 days after the challenge and analysed with confocal microscopy for GL7 (red) and B220 (blue) expression on expanding memory GFP+ (green) B cells. (e) Close-ups of the PP section marked in the mid panel in d with each individual channel shown in black and white and an overlay in colour in which individual GFP+ cells are circled to illustrate that most responding GFP+ memory B cells express B220, but not the GL7 marker even if located in a germinal centre. (f) Flow cytometry analysis on day 7 of IgA expression in responding GFP+ memory B cells in the PP and MLN. The percentage of IgA+ B cells/all GFP+ B cells in the PP or MLN in individual mice is given (right panel). (g) Flow cytometry analysis of the expression of CD73 and PD-L2 in responding GFP+ CD19+B220+CD138− memory B cells on different days following a p.o. challenge immunization. Each bar represents the percentage of memory B cells that co-express CD73 and PD-L2 (grey) or only CD73 (black). (h) Mice p.o. primed with NP-CT received a p.o. or i.p. challenge immunization 6 months later in the presence or absence of anti-CD40L Mab (clone MR-1), and the proportion of GFP+ cells/total B220+ B cells or CD138+ plasma cells in the lamina propria (LP), spleen (Spl) or PP in individual mice was determined using flow cytometry. The Mann–Whitney non-parametric test showed that GC disruption with anti-CD40L Mab significantly reduced GFP+ plasma cell numbers in the SI LP (P=0.0193) and responding B-cell numbers in the PP (P=0.0053). (i) NP-specific GFP+ cells in the PP or spleen in p.o. or i.p. challenged mice were divided into responding memory B220+ B cells and GFPhigh B220− plasmablasts/plasma cells (shown in Supplementary Fig. 6e,f), and the expression of IgA, CCR9, CCR10 and integrin α4β7 was determined by flow cytometry. As a control, GFP− B cells from the PP are included in each panel. (j) The proportion of mice that express CCR9 or IgA of the indicated GFP+ cell populations after an i.p. (open symbols) or p.o. (closed) challenge immunization. Significance was calculated with the Mann–Whitney test (f) and analysis of variance followed by Tukey's multiple comparison correction (j) and P values are given (**0.005≤P<0.01, ***0.001≤P<0.005, ****P<0.001). Data from four to five mice in each group in one representative experiment out of three giving similar results are shown.

Fig 5: Selection of broadly reactive IgG is regulated differently from strain-specific IgG.a B220+IgM−IgD−Dump− B cells were obtained from spleen of the mice immunized with inactivated A/Narita/1/2009 (Vaccine, white bars; n = 3) and from MLN in the A/Narita/1/2009 infected mice (Infection, black bars; n = 3). Binding to A/Narita/1/2009-HA and/or A/PR/8/1934-HA was determined by flow cytometry analysis. b The data indicate binding to HA in flow cytometry analysis (left) and the number of single or dual-binding B cells (right) in spleen (white bars) or MLN (black bars) from the mice infected with A/Narita/1/2009 (n = 5). c Narita-HA binding B cells (2000 cells each) were isolated from vaccinated mice (spleen, white bar; n = 3) and infected mice (MLN, black bar; N = 3). The V-D-J combinations are shown by connected color bands between V and D (left), V and J (middle), and D and J (right). d Vh gene usage of A/Narita/1/2009-HA binding B cells in spleen from three vaccinated mice (Vaccine n = 3) or MLN (Infection n = 2) from two infected mice with A/Narita/1/2009. Total RNA isolated from sorted A/Narita/1/2009-HA binding B cells served as the template for cDNA synthesis followed by PCR to amplify IgG variable region genes. Amplified IgG gene libraries were sequenced by MiSeq. The IgG sequences were analyzed with the ImMunoGeneTics (IMGT) HighV-QUEST. e Seven hundred twenty HA probe+ B cells were single sorted and cultured on NB21.2D9 feeder cells with IL-4 in individual wells. After 20 days of culture, 303 IgG-producing cells were obtained. The binding of antibodies produced by these cells to A/Narita/1/2009-HA, A/PR/8/1934-HA, A/H1N1pdm09 derived LAH (blue), or rHA2 (red) was measured by ELISA. Finally, reliable binding and VH genetic information were obtained for 101 clones. Black circles indicate clones showing no binding to LAH or rHA2. Y-axis represents the ratio of the OD450 value in the sample vs the OD450 value obtained with IgG [anti-Narita-HA (1 µg/ml), anti-PR8-HA (1 µg/ml), anti-LAH (1 µg/ml), and rHA-2 (3 µg/ml)]. f Amino acid sequences encoded by Vh genes in single or dual-HA binding B cell clones in the region from CDR1 to CDR3 are shown with germline amino acid sequence (unhighlighted). Red boxes indicated CDR regions. The positions where amino acid substitutions occur are indicated by the red font. g Number of amino-acid mutations in the region extending from CDR1 to CDR3 of single or dual-binding B cell clones (single, blue dots; n = 18, dual, red dots; n = 31) as determined by referring to the germline sequences of the corresponding Vh in the IMGT database. *p < 0.05, **p < 0.01 by unpaired, two-tailed t-test (Vaccine group with Infection group in a and c, Spleen group with MLN group in b, Single group with Dual group in g). All error bars represent SEM.