Mo-DCs are the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59.

<p>Popliteal dLN cell suspensions from mice immunized with PE or PE + MF59 analyzed by flow cytometry 18 hrs after immunization. (<b>a</b>) Upper panels: fow cytometry dot plots that show the uptake of PE and MF59 by MΦs (blue cells), DCs (green cells) and monocytes or Mo-DCs (red cells) from one representative experiment in mice immunized with PE or PE + fluorescently labelled-MF59 are reported as example. Middle and lower panels: bar graph histograms report the percentage (middle panels) and the number (per million of live/singlet cells acquired by flow cytometry) (lower panels) of PE positive cells in MΦs (blue bars), DCs (green bars) and monocytes or Mo-DCs (red bars). Data from three independent experiments (single numbered bars) and the arithmetic mean of these values (black horizontal line) are shown. Statistical analysis: parametric one–way ANOVA test (Dunnett’s multiple comparison using Mo-DCs as control column) has been applied to calculate the P-value.<i>*P</i>˂0.05; <i>***P</i>˂0.001; <i>****P</i>˂ 0.0001. (<b>b</b>) Bar graph histogram reports the average (+ standard deviation) number (per million of live/singlet cells acquired by flow cytometry) of PE positive MΦs (blue bars), DCs (green bars) and Mo-DCs vs. monocytes (red bars) in mice immunized with PE (dotted bars) or PE + MF59 (filled bars) in the three independent experiments reported in panel (a). Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. <i>*P</i>˂0.05; **<i>P</i><0.01. (<b>c</b>) Upper panels: flow cytometry histograms of CD80 expression by MΦs (blue), DCs (green) and Mo-DCs vs. monocytes (red) in mice immunized with PE (thin lines) or PE + MF59 (thick lines) from one experiment out of the three shown in panel (a). Lower panel: bar graph histogram shows the difference in CD80 Mean Fluorescence Intensity (MFI) of MΦs (blue bars), DCs (green bars) and Mo-DCs vs. monocytes (red bars) from mice PE immunized with and without MF59 (ΔMFI). Data of the same three independent experiments reported in panel (a) (single numbered bars) and the arithmetic mean of these values (black horizontal line), are shown. Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. <i>**P</i>˂0.01.</p

Simultaneous identification of B lymphocytes specific for HA from different influenza strains in <i>ex vivo</i> PBMCs samples.

<p><b>A.</b> PBMCs from an anonymous blood donor were pre-incubated with subunits from either B/Brisbane/60/2008 or A/Panama/2007/1999 (H3N2), and then stained with anti-CD20 mAb, HSA conjugated with A488 and A647, with A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09), or with A647-rB/HA (from B/Brisbane/60/2008) and A488-rH1 (from A/California/07/09). The staining patterns observed in the CD20⁺ B-cell gate are shown. <b>B.</b> PBMCs from 16 anonymous blood donors were pre-saturated with B/Brisbane/60/2008 and then stained with anti-CD20 mAb, A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09). The scatter plot depicts paired values of H1⁺ (y-axis) and H3⁺ (x-axis) B-cells. The insert box plot depicts the distribution of H1⁺, H3⁺ and H1⁺H3⁺ B-cells in the same 16 donors. Mean values are indicated by dotted lines.</p

MF59 promotes differentiation and transient accumulation of Mo-DCs within the dLN.

<p>(<b>a</b>) Left panel: flow cytometry dot plots of dLN cell suspensions to identify APCs are reported for each time point and each treatment (as indicated). Macrophages (MΦ) are depicted in blue, whereas the rest of the APCs are in black. The transient appearance of a CD8α_F4/80 double positive APC subset (red gates) between 8 hrs and 18 hrs after immunization (red quadrant) is shown. Representative results of one experiment out of three are shown. Right panel: bar graph histogram that reports the number x10³ (per million of live/singlet cells acquired by flow cytometry) of CD8α_F4/80 double positive APCs at 8 hrs and 18 hrs after immunization with PE (white bars) and PE + MF59 (grey bars). Data from three independent experiments (single numbered bars) and the arithmetic mean of these values (black horizontal line) are shown. Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. **<i>P</i><0.01. (<b>b</b>) Flow cytometry dot plots of dLN cell suspensions obtained 18 hrs after immunization with PE or PE + MF59 are shown to analyze the expression of the surface protein CD64 on the different APC subsets. CD8α_F4/80 double positive APCs are depicted red, LN DCs in green and MΦs in blue. Representative results of one experiment out of the three independent experiments reported in panel (a) are shown. (<b>c</b>) Flow cytometry dot plots of dLN cell suspensions obtained 8 hrs and 18 hrs after immunization with MF59 reveal that the CD8α_F4/80 double positive APC subset (red cells) expresses the CD11c DC marker 18 hrs but not 8 hrs after the immunization. Representative results of one experiment out of the three reported in panel (a) are shown. (<b>d</b>) Flow cytometry dot plots of the dLN cell suspensions to identify APCs derived from mice immunized with MF59, whose LNs were explanted from both legs 8 hrs after the immunization, but: the LNs from the left legs (left dot plots) were immediately processed (8 hrs), whereas the LNs from the right legs (right dot plots) were processed after 10 hrs of in vitro culture (8 hrs + 10 hrs (in culture)). Representative results of one experiment out of three are shown.</p

H1⁺ IgG⁺ MBCs frequencies measured by flow-cytometry and by ELISPOT correlated linearly.

<p><b>A–B.</b> Specificity of the staining with the rH1 bait from A/Solomon Island/3/06. PBMCs (1.6×10⁸) from anonymous blood donors were stained with Live/Dead, incubated with an H3N2 mono-bulk vaccine subunit (from A/Panama/2007/1999), and then stained with Alexa647-conjugated HSA (6×10⁷), or Alexa647-conjugated rH1 (1×10⁸), and with an antiCD20 mAb. <b>A.</b> Binding pattern of HSA (A647-HSA; left panel) and of rH1 (A647-rH1; right panel) in the CD20⁺ B-cell gates. <b>B.</b> H1⁺ B-cells identified in A were sorted (n = 8215), mixed with autologous CD20^neg cells in the ratio of 1∶50 and activated with CpG and IL-2 for 5 days <i>in vitro</i>. Unsorted PBMC and CD20^neg cells were also cultured in the same manner as controls. After 5 days, equal numbers of cultured cells were harvested and assayed by ELISPOT for numbers of cells secreting IgG and IgG specific for H1N1 (from A/Solomon Island/3/06). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#s2" target="_blank">Results</a> are expressed as number of antibody secreting cells (ASC) normalized to 10⁶ cultured cells assayed by ELISPOT. Nd indicates undetectable ASC. <b>C.</b> Distribution of IgG⁺ B-cells among H1^neg and H1⁺ B cells expressing or not the CD27 B cell memory marker; shown is one representative subject. <b>D.</b> Replicates of frozen PBMCs from 4 anonymous blood donors were assayed by conventional ELISPOT, or incubated with an H3N2 mono-bulk vaccine subunit and stained with rH1, and anti-CD20 plus anti-human IgG antibodies. The scatter plot depicts paired values of H1⁺ IgG⁺ B-cell frequencies measured by flow-cytometry (y-axis) and by ELISPOT (x-axis) across three different experimental sessions. Shown are: the regression line with the related 95% confidence interval (gray areas), slope, intercepts, R² and p-value. <b>E.</b> Variability plot showing mean standard deviations of the measurements done by ELISPOT and flow-cytometry. The three dotted lines mark the grand mean and the upper and lower control limits.</p

Identification of B lymphocytes specific for HA from A and B influenza strains in <i>ex vivo</i> PBMCs samples.

<p>PBMCs from different anonymous blood donors were pre-incubated with vaccine mono-bulk subunits from the B/Brisbane/60/2008 (B/HA pretreatment), or the H3N2 A/Panama/2007/1999 strain (A/HA pretreatment) and then stained HSA, rH3 (from A/Brisbane/10/2007), rH1 (from A/California/07/2009), or B/HA (from B/Brisbane/60/2008), as indicated. <b>A.</b> Staining pattern observed on CD20⁺ cells in PBMCs stained with the different rHA bait. The rectangular gates identify brilliant HA+ B-cells; the dotted vertical lines mark the gates used to sort HA⁺ B-cells for the ELISPOT assays. <b>B.</b> Expression of the CD27 memory marker on HA⁺ and HA^neg B cells identified based on the sorting gates. <b>C.</b> H3⁺ (n = 15,234), H1⁺ (n = 6482) and B/HA⁺ (n = 26,803) B-cells identified in A were sorted, mixed with autologous CD20^neg cells (in the ratio of 1∶20, 1∶100 and 1∶33) and activated with CpG and IL-2 for 5 days <i>in vitro</i>. Unsorted PBMCs and CD20^neg cells mixed with HA^neg B cells were also cultured in the same manner, as controls. After 5 days cultured cells were harvested and assayed by ELISPOT for the number of cells secreting IgG and IgG specific for mono-bulk subunits from the vaccine strain homologous to the sorting bait. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#s2" target="_blank">Results</a> are expressed as numbers of antibody secreting cells (ASC) normalized to 10⁶ cultured cells assayed by ELISPOT. Nd indicates undetectable ASC.</p

Molecular cloning of HA⁺ B lymphocytes.

<p>PBMCs from 4 anonymous blood bank donors were stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#pone-0070620-g004" target="_blank">Figure 4B</a>. Single H1⁺, H3⁺, or H1^negH3^neg CD20⁺ B-cells were sorted to perform molecular cloning and analysis of their paired V_HV_L Ig regions as described in Material and Methods section. <b>A–E.</b> Distribution of V_H (A), D_H (B), J_H (C), V_k (D) and J_k (E) gene use across arrays of B-cells sorted from each donor (16 and 18 HA⁺ clones from donors #1 and #2; 35 HA⁺ and 20 HA^neg clones from donor 3; 16 HA⁺ and 16 HA^neg clones from donor #4. <b>F–I.</b> Number of mutations in H1⁺ and H1^neg CD20⁺ B-cells from donors #3 and #4, which cause dissimilar (F, H) or similar (G, I) amino acid substitutions in V_H (G,I) and V_L (F,H). NS and ** indicate not significant, or significant (p<0.036) difference between mean numbers of mutations by one-way Wilcoxon non-parametric test.</p

Vaccination induced changes in the pool of H1+ B-cells.

<p>PBMCs samples collected before (day 0) and at 3 and 6 weeks after vaccination from four seasonal influenza vaccinees, were pre-incubated with H3N2 subunit (from A/Panama/2007/1999) and then stained with rH1 A/Solomon Island/3/06) and mAbs anti-CD20, anti-CD27 and anti-human IgG. <b>A.</b> Dot plots gated on CD20⁺ B-cells showing the distribution of H1⁺ (middle panels) and H1^neg (bottom panels) B-cells from donor #a across: the mature memory (CD27⁺) and putatively naive (CD27^neg) CD20+ B-cell subsets (upper panels); un-switched mature memory (CD27⁺IgG^neg), IgG-switched mature (CD27⁺IgG⁺) and immature (CD27^negIgG⁺) memory B-cells. <b>B.</b> Numbers of circulating H1⁺ CD20⁺ B-cells in 4 vaccinees before and at 3 and 6 weeks after seasonal vaccination are overlaid with paired titers of antibodies inhibiting virus-induced hemmaglutination measured in their blood. The frequencies of H1⁺ B-cells are normalized according to the frequencies of CD20⁺ B-cells in 10⁶ PBMCs. <b>C.</b> Distribution of circulating H1⁺ and H1^neg B-cells across same subsets identified in <b>B</b> in all vaccinees.</p

Co-localization of antigen, MF59 and DCs within the medullary compartment of the dLN.

<p>(<b>a</b>) Confocal microscopy images of dLNs collected from mice 18 hrs after treatment with PBS, PE or PE + MF59 and labeled to detect DCs and the medullary compartment of the LN. Signals which detect the LN medulla (blue, F4/80), PE (red) and DCs (yellow, CD11c) are shown separately and merged (as indicated). The magnification shows the co-localization of antigen and DCs within the LN medulla. The image of one section is shown, in each panel, as example of consecutive sections of a whole LN, which is representative of the organs of all immunized mice. Original magnification: 5X. Scale bar: 1 mm. Results of one representative experiment out of three are reported. (<b>b</b>) Confocal microscopy images of dLNs collected from mice 18 hrs after treatment with PE + fluorescently labelled-MF59 and labeled to detect DCs and the medullary compartment of the LN. Signals which detect the LN medulla (blue), MF59 (green), PE (red) and DCs (yellow) are shown separately and merged (as indicated). The magnification shows the co-localization of antigen, MF59 and DCs within the LN medulla. The image of one section is shown, in each panel, as example of consecutive sections of a whole LN, which is representative of the organs of all immunized mice. Original magnification: 5X. Scale bar: 1 mm. Representative results of one representative experiment out of two are reported.</p

Magnitude and quality of vaccine-specific CD4⁺ T-cell responses induced by 4C-Staph/T7-alum and 4C-Staph/alum.

<p>Splenocytes from single mice (n = 16) vaccinated with 4C-Staph/T7-alum, 4C-Staph-alum, T7-alum or alum by 12 days were stimulated or not with vaccine antigens <i>in vitro</i>, stained and analyzed by intracellular cytokine staining. CD4⁺CD44^high T cells producing IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A were identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147767#pone.0147767.s001" target="_blank">S1 Fig</a> for gating strategy). The response of unstimulated cells was subtracted from that of stimulated cells. Data are the merge of four independent experiments. (<b>A</b>) Percentages of CD4⁺CD44^high T cells producing any combination of IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A in response to vaccine protein stimulation (CD4⁺CD44^high total CYT⁺ cells) were calculated applying Boolean gates. Bars represent mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by one-way ANOVA and Sidak post-test. (<b>B</b>) Percentages of CD4⁺CD44^high T cells producing IL-2, TNF, IL-4/IL-13, IFN-γ and/or IL-17A in each of the possible combinations (CD4⁺CD44^high CYT⁺) in response to vaccine proteins stimulation calculated applying Boolean gates. No cells expressing more than 3 cytokines at once were detected. Bars represent mean ± SEM. *<i>p</i> < 0.05 by unpaired Student <i>t</i> test, two-tailed, and a partial permutation test.</p

One dose of 4C-Staph/T7-alum induces functional antibodies.

<p>BALB/c mice (n = 16) were immunized once with 4C-Staph/T7-alum or 4C-Staph/alum. Control mice were injected with T7-alum or alum alone. (<b>A</b>) Vaccine-specific serum IgG titers measured 12 (d12) and 32 (d32) days after vaccination. IgG concentrations in control sera (open symbols) are reported only for d32. Each symbol represents one mouse, and data are the merge of two independent experiments. Median with interquartile range of each group is also shown. *<i>p</i> < 0.05, ****<i>p</i> < 0.0001 by Kruskal-Wallis test and Dunn's multiple comparisons test. (<b>B</b>) Hla neutralizing activity of pooled sera from vaccinated mice (n = 16, same animals as in A) was assessed on rabbit RBCs and expressed as effective dilution that neutralized 50% of Hla lytic activity (ED₅₀). No hemolysis inhibition was detected (ED₅₀ < 6) in pre-immune sera or in sera from adjuvant-treated mice. Lack of overlap in the 95% confidence intervals between the ED₅₀ of sera from mice vaccinated with 4C-Staph/alum (30.3 to 51.4) vs. 4C-Staph/T7-alum (114.6 to 318.7) by 32 days indicates a difference significant with <i>p</i> < 0.05. Bars represent SEM. (<b>C</b>) Vaccine-specific IgG1 and IgG2a. Columns represent median MFI with interquartile range of pooled sera from vaccinated mice (n = 16, same pools as in B) bled at d32. **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by unpaired Student <i>t</i> test, two-tailed. (<b>D</b>) Hla_H35L-specific IgM. Columns represent median MFI with interquartile range of sera from vaccinated mice (n = 12) bled at d12. IgM specific for EsxAB, FhuD2 and Csa1A were at the limit of detection (data not shown). Data shown are the merge of two independent experiments.</p