Fig 1.
Experimental designs and readouts.
Adult blood (AB) and cord blood (CB) samples were stimulated by different B. pertussis isolates (FR4930, FR5730, FR5862, FR5333, FR6440 or Tohama) at concentrations ranging from 1.75x107 to 4.2x108 CFU/mL for 4h or 22h. Cytokine release was analyzed using Luminex assays, immune cell profiling was performed by flow cytometry, and transcriptomic analysis was conducted by NanoString Technologies. Figure created in BioRender. Sadi, M. (2025) https://urldefense.com/v3/__https://BioRender.com/ctfvluo__;!!JFdNOqOXpB6UZW0!s9oPd_cZWW4YU9Tj_YyjedOUqXppKnb2gOy6VkBy9GgZkyi2Wl5433Uko5wi9ise2apVl_HrqzyrJ8vuKkTAxFg$.
Fig 2.
Analyses of cytokine/chemokine levels in plasma samples.
Cord blood (CB, n = 8) and adult blood (AB, n = 9) from healthy donors were stimulated ex vivo with B. pertussis (isolate FR4930, 1.75x107 to 4.2x108 CFU/mL). Analytes were measured in whole blood plasma/supernatant after 22 hours of infection using a 27-analytes panel (Human Luminex Assay R&D Systems, Minneapolis). (A) Raw data of concentrations (in pg/ml) of 27 cytokines, chemokines and growth factors measured on plasma from AB (blue plots) and CB (red plots) stimulated with B. pertussis (FR4930) for 22 hours. Non stimulated plasma samples were used as controls (in black). Levels are represented in median and interquartile. (B) The heatmap displayed all analytes differentially secreted by stimulated CB (in red) compared to stimulated AB (blue) samples (adjusted p-value < 0.05). In heatmap (B) cytokines/chemokines are expressed in pg/mL and log transformed, with blue to red colors representing lower to higher secretion respectively. On the x-axis, experiments are organized by blood type (blue, AB; red, CB) and by concentration levels of B. pertussis (left to right, from 1.75x107 to 4.2x108/ml CFU/mL). On the y-axis, cytokines/chemokines are displayed following hierarchical clustering. Heatmap were created using Qlucore OMICS explorer 3.7. (C) Levels (in pg/mL) of VEGF-A, IL-1α, IL-1β, IL-12p40, TNFα and IFNγ in AB (blue) and CB (red) are displayed according to the levels of B. pertussis used for stimulation (from 0 to 4.2x108 CFU/mL). Each dot/square represents the median level of the cytokine according to the appropriate range of CFU/mL with its interquartile.
Fig 3.
Characterization of cell populations in CB at baseline and after a 4-hour stimulation with B. pertussis.
(A) Concatenated t-distributed stochastic neighbor embedding (t-SNE) analysis of CB samples to identify 12 distinct immune cell types based on the expression of phenotypical markers by spectral flow-cytometry. Erythroid progenitors were added to the analysis in a separated spectral flow-cytometry panel. (B) Relative frequency distribution of the different immune cell types at baseline in CB as compared to AB. (C) Representative flow cytometry plots of CD66b and CD16 expression by granulocytes from whole-blood, ex-vivo or after 4h at 36°C, 5% CO2 in the absence (non-stimulated, NS) or presence of B. pertussis (isolate FR4930). Dot-plot representation (with median) of the Mean Fluorescence Intensity (MFI) for the expression of CD66b and CD16, across the different experimental stimulation conditions, as well as across CB (N = 4) and AB (N = 6). (D) Correlation analysis to assess the relationship between cell count and cell frequency across the different immune cell types from AB and CB, after 4-hour stimulation with B. pertussis (FR4930) and as compared to baseline (NS). Each point represents the median of relative cell count and cell frequency for the different immune cell types. (E) Dot plot histograms displaying the cell frequency of mMDSCs in CB across the different experimental conditions. (F) Linear regression analysis to model the relationship between the loss of detection of mMDSCs (expressed as Fold Changes as compared to ex-vivo) and the inoculum concentration of different B. pertussis isolates (FR4930, FR5730 and FR5333, expressed in CFU/mL).
Fig 4.
Characterization of cell populations in CB after a 22-hour stimulation with B. pertussis.
(A) Correlation analysis to model the relationship between cell count and cell frequency across the different immune cell types from AB and CB, after 22h stimulation with B. pertussis (FR4930). Each point represents the median of relative cell count and cell frequency for the different immune cell types after stimulation, when compared to baseline control (22-hour incubation NS). (B) Dot plots showing the frequency of CD25 ⁺ B cells in adult blood (AB, blue) and cord blood (CB, red) across experimental conditions. Four B. pertussis isolates (FR4930, FR5730, FR5333, and Tohama) and a reference E. coli strain (S88) were tested. Sample sizes (AB/CB): ex vivo (11/14), non-stimulated (11/14), FR4930 (6/7), FR5730 (5/5), FR5333 (3/4), Tohama (1/3), E. coli (3/4). For statistical analyses, all B. pertussis-stimulated samples were pooled into a single group (AB, n = 18; CB, n = 23), regardless of isolate. (C) After 22 hours of stimulation with B. pertussis (FR4930 isolate), cells were all stained for CD3, CD19, CD25 and for either CD27, CD21, CD5, CD10, CD71 (1st B-cell panel), or for CD24, CD38, CD40, CD80, IgD, IgM and IgG (2nd B-cell panel), and analyzed by flow cytometry. CD19 + CD25-negative cells were represented in blue, CD19 + CD25 + cells were represented in red, and total live cells in grey.
Fig 5.
Genes differentially regulated in CB after a 4-hour stimulation with B. pertussis.
(A) Heatmap representing differentially expressed genes (log2FC ± 1.5, adjusted p-value < 0.05) in cord blood (CB) stimulated ex vivo with different B. pertussis isolates (FR4930, FR5730, FR5862, FR6440) compared to NS CB conditions. On the x-axis, blood donors are organized by blood type, the experiment (n = 3 independent experiments, corresponding to 3 different donors for each blood type) and the B. pertussis isolate used for ex vivo stimulation. The y-axis corresponds to each gene displayed following hierarchical clustering. The heatmap visualizes normalized gene expression values scaled per gene by centering the mean and dividing by the standard deviation, providing an overview of gene expression patterns. Blue to red colors represent lower to higher expression respectively. (B) Gene expression pathways enriched in CB stimulated with B. pertussis. The bar plots depict pathways that are significantly enriched (dark red, -logit(p-value) > 2.5) or diminished (blue). Pathways are listed on the y-axis, while the x-axis represents the corresponding -logit(p-value).
Fig 6.
Genes differentially regulated between CB and AB after a 4-hour stimulation with B. pertussis.
(A) Heatmap representing the genes with differential slopes (log2FC=0, adjusted p-value < 0.05) between AB and CB, where each slopes reflects the expression trajectory between no stimulation (NS) and stimulation conditions. On the x-axis, blood donors are organized by blood type, the experiment (n = 3 independent experiments corresponding to 3 different donors for each blood type) and the B. pertussis isolate used for ex vivo stimulation. The y-axis corresponds to each gene displayed following hierarchical clustering. The heatmap visualizes normalized gene expression values scaled per gene by centering the mean and dividing by the standard deviation, providing an overview of gene expression patterns. Blue to red colors represent lower to higher expression respectively. (B) Boxplots representing upregulated genes in AB, as compared with CB (changes in gene expression in response to infection were compared, adjusted p-value < 0.05). (C) Boxplots representing downregulated genes in AB, compared to CB (adjusted p-value < 0.05). (D) Gene expression pathways enriched in CB compared to AB, upon stimulation with B. pertussis. The bar plots depict pathways that are significantly enriched (dark red, -logit(p-value) > 2.5) or diminished (blue). Pathways are listed on the y-axis, while the x-axis represents the corresponding -logit(p-value).
Fig 7.
Gene expression in isolated B cells derived from CB.
Heatmap representing differentially expressed genes (log2FC ± 1.5, adjusted p-value > 0.05) in B cells upon stimulation with B. pertussis FR4930. CD25 + B cells stimulated for 22h are compared to non-stimulated CD25+ and CD25- B cells. On the x-axis, the isolated B cells are organized according to stimulated (S) or non-stimulated (NS) CD25- or CD25 + B cells. The data is representative of three independent experiments. The y-axis corresponds to each gene displayed following hierarchical clustering. The heatmap visualizes normalized gene expression values scaled per gene by centering the mean and dividing by the standard deviation, providing an overview of gene expression patterns. Blue to red colors represent lower to higher expression respectively.