Phenotypic properties of hIRA B cells from tonsils.

<p>Conventional CD19⁺CD20⁺GM-CSF^-(GM-CSF^neg) and hIRA CD19⁺CD20⁺GM-CSF⁺(GM-CSF^pos) B cells in cell suspensions from tonsils were analyzed by flow cytometry. A) Expression of CD5, CD43, CD11c and IgD in GM-CSF^neg versus GM-CSF^pos cells. The graphs show the geometric mean of fluorescence intensity (GeoMFI) for each marker after isotype background subtraction. Means and standard deviations are shown. P value was calculated using the non-parametric Mann–Whitney test (n = 4). B) Ig subclasses were analyzed within the hIRA B cell population. The plots are representative of n = 4.</p

hIRA B cells are present in human tonsils.

<p>A) Gating strategy for the identification of hIRA B cells in tonsils. Tonsil-derived cells were obtained from tonsils of individuals undergoing routine tonsillectomy. After processing in the presence of DNAse and collagenase, samples were stained, fixed and analyzed by flow cytometry. Live cells were discriminated using Aqua Live/Dead staining kit, and singlet lymphocytes were selected on the basis of forward (FSC) and side (SSC) scatters. B cells were further identified as CD19⁺CD20⁺ and analyzed for the expression of GM-CSF. B) Quantification of hIRA B cells in tonsils. The number of CD19⁺CD20⁺GM-CSF⁺ cells was normalized on the number of total CD19⁺CD20⁺ lymphocytes after isotype background subtraction. The bar represents the geometric mean.</p

Effect of different stimuli on the frequencies of CD19⁺CD20⁺GM-CSF⁺ cells.

<p><i>Ex-vivo</i> cells from tonsils (5 x 10⁵ cells/well) were stimulated for 2 days at 37°C in the presence of IL-2 with or without polyclonal stimuli (anti-IgM 10 μg/ml or SAC 1:20.000 final) or TLR9 ligand (CpG at 10 μg/ml). Brefeldin A was added 3 h before fixation and staining.</p

Human IRA B cells reside within the follicles.

<p>A) Cell suspensions from tonsils were seeded onto poly-L-lysine coverslips and fixed with formaldehyde. Cells were incubated for 1 h with anti-CD19 (Green) and anti-GM-CSF (Red). The merge panel shows the co-localization of GM-CSF and CD19. The picture is representative of three different subjects. Scale bar = 10 μm. B) 8-μm tonsil tissue sections were fixed with formaldehyde and stained using anti-CD3 (T cell area, Blue), anti-CD19 (B cell area, Green) and anti-GM-CSF (Red). The enlargement shows the presence of GM-CSF⁺ cells within B cell follicles (white arrows). The panel is representative of 3 independent experiments using different tonsils where many sequential sections were screened (n>5). Scale bar = 50 μm. C) 8-μm tonsil tissue sections were fixed with formaldehyde and stained using anti-CD3 (T cell area, Blue), anti-IgD (mantle zone, Green) and anti-GM-CSF (Red). The panel is representative of 3 independent experiments using different tonsils where many sequential sections were screened (n>5). Scale bar = 50 μm.</p

hIRA B cells have phagocytic capacity.

<p>A) Tonsil-derived cells were incubated for 2 h at 37°C with heat-inactivated <i>S</i>. <i>aureus</i> at 1:10 (cells:bacteria) MOI. After staining with anti-CD19 (Blue), anti-GM-CSF (Orange), and anti-MPO (Red), different optical fields were analyzed by confocal microscopy. The enlargement (bright field merged with the GM-CSF channel, right panel) shows the presence of <i>S</i>. <i>aureus</i> internalized by a CD19⁺GM-CSF⁺MPO⁺ cell (white arrows) and two CD19⁺GM-CSF^-MPO^- cells with non-internalized bacteria (yellow arrows). B) <i>Left panels</i>: similar experimental approach described for panel A) with the addition of PhRodo labelled <i>S</i>. <i>aureus</i> bioparticles (green). The white arrows indicate not-internalized bioparticles (low fluorescence intensity), while bright fluorescent bioparticles localize within a CD19⁺GM-CSF⁺MPO⁺ cell (white square) but not within a conventional CD19⁺ cell (yellow square); <i>right panels</i>: control experiment where cells were incubated with PhRodo labelled <i>S</i>.<i>aureus</i> bioparticles (dark green) for 2 h at 4°C. Data are representative of 3 independent experiments. Scale bar = 10 μm.</p

H1N1-specific CD4⁺IL-21⁺ICOS⁺CXCR5⁺ T_FH cells predict functional antibody responses.

<p>Correlations between the number of H1N1-specific CD4⁺IL-21⁺ICOS⁺CXCR5⁺ T_FH cells and H1N1-specific DHI responses measured at (A) day 28 and (B) day 168 after immunizzation. Dashed lines represent the least squares regressions fit to the data. R: Pearson product-moment correlation coefficient. <i>p</i>: correlation-associated p value.</p

Expansion of ICOS⁺ and PD-1⁺ T_FH1 cells after TIIV and ATIIV vaccination.

<p>(<b>A</b>) Number of CD4⁺ T cells expressing CXCR5 and ICOS in human PBMCs after seasonal influenza vaccination. (<b>B</b> and <b>C</b>) Number of T_FH1 cells expressing ICOS and PD-1. Data show three cohorts: saline placebo (n=7), TIIV (n=18) and ATIIV (n=17) at baseline (D0), day 7 (D7) and day 28 (D28) after a single dose of influenza vaccine. Data are shown for each participant and expressed as number of cells in 10⁶ live PBMCs acquired. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared to day 0; ^§<i>p</i> < 0.05, ^§§<i>p</i> < 0.01 and ^§§§<i>p</i> < 0.001 compared to saline placebo.</p

T_FH1 ICOS⁺ cells predict functional antibody responses.

<p>Correlations between the number of total circulating CD4⁺ T_FH1 ICOS⁺ cells and the maximum DHI responses observed across the three influenza strains represented in the vaccine, measured at (A) day 28 and (B) day 168 after immunizzation. Dashed lines represent the least squares regressions fit to the data. R: Pearson product-moment correlation coefficient. <i>p</i>: correlation-associated p value.</p

H1N1-specific CD4⁺IL-21⁺ICOS⁺ T_H cells subsets expressing or not CXCR5 expand after influenza vaccination.

<p>Number of CD4⁺IL-21⁺ICOS⁺ T_H cells, showing a CXCR5⁺ (black) or CXCR5^- (gray) phenotype, in vaccinated participants after overnight stimulation with A/California/7/2009 (H1N1) antigen or SEB. Data show saline placebo (n=7), and merged TIIV (n=18) and ATIIV (n=17) cohorts at baseline (D0), day 7 (D7) and day 28 (D28) after a single dose of influenza vaccine. Data are expressed as number of cells in 10⁶ live CD4⁺ T cells; mean ± SEM is shown. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared to day 0.</p

B-cell and functional antibody responses after seasonal influenza vaccination.

<p>(<b>A</b>) Absolute number of plasmablasts (CD19⁺CD20^- CD38⁺) in 10⁶ live PBMCs acquired. (<b>B</b>) HI Geometric mean titers (GMT) for A H1N1 and H3N2, and B influenza strains at baseline (D0), 7 days (D7), 28 days (D28), and 168 days (D168) after a single dose of influenza vaccine. Data show three cohorts: saline placebo (n=7), TIIV (n=18), and ATIIV (n=17). (<b>C</b>) Geometric mean ratio (GMR) for all vaccine strains. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared to day 0; ^§<i>p</i> < 0.05, ^§§<i>p</i> < 0.01 and ^§§§<i>p</i> < 0.001 compared to saline placebo.</p