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

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

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