Immunology and Inflammation

T follicular helper cell profiles differ by malaria antigen and for children compared to adults

Catherine S. Forconi
Christina Nixon
Hannah W. Wu
Boaz Odwar
Sunthorn Pond-Tor
John M. Ong’echa
Jonathan Kurtis
Ann M. Moormann author has email address

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA
Center for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya

https://doi.org/10.7554/eLife.98462.1

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Figures and data

Pf-malaria IgG sero-profiles for children and adults.
(a) IgG antibody levels against AMA1, MSP1, HRP2, CelTos and CSP for children (n=14). The bar plots indicate mean with SD. (b) IgG antibody levels against PfSEA-1A and PfGARP comparing children (n=15) and adults (n=15). The bar plots indicate mean with SD. Net MFI values are the antigen-specific MFI values minus the BSA background. Mann-Whitney tests were performed.

Clusters visualization of immune cell types and their abundance.
(a) A 100 node and 75-meta-clusters FlowSOM tree was generated on live lymphocytes from all our participants (n=29 children and adults), highlighting CD3^posCD8^pos, CD3^posCD4^pos, CD4^posCXCR5^pos and CD4^posCD25^pos cells. The 5 nodes of the CD4^posCXCR5^posCD25^neg population are circled and will be used for the downstream analysis. The colored scale was based on the median arcsinh-transformed marker expression. UMAP plots showing 5 clusters defined as follow: CD14/CD19^pos (orange), CD4^pos (blue), CD4^posCXCR5^pos (red), CD8^pos (purple) and CD4^posCD25^pos (green) from (b) adults (n=15) (c) children (n=14) PBMCs after no-stimulation, PfSEA-1A and PfGARP stimulation, respectively from left to right.

Characterization of T_FH subsets by CXCR5, CXCR3, CCR6 and CD45RA expression.
(a) A new 25 nodes and 15 meta-clusters FlowSOM tree was generated from the 5 CD4^posCXCR5^posCD25^neg nodes shown in Figure 2A (CXCR5 expression tree). The colored scale of CXCR5 expression was based on the median arcsinh-transformed with red as the highest and deep blue the absence of CXCR5 expression. (b) Box-plots showing CXCR5 expression across all cT_FH-like meta-clusters from children (n=13) with no stimulation (blue), and after in vitro stimulation with PfGARP (green) or PfSEA-1A (pink). Fifty percent of the data points are within the box limits, the solid line indicates the median, the dash line the mean and the whiskers indicate the range of the remaining data with outliers being outside that range. Similar box-plots are shown for (c) CD45RA, (d) CXCR3, and (e) CCR6 expression across all cT_FH-like meta-clusters. (f) Clustered heatmap showing the median arcsinh-transformed expression for CCR6, CD45RA, CXCR3 and CXCR5 across meta-clusters, red showing the highest expression and blue the lowest. (g) Cytoplots of CCR6 vs CXCR3 expression where MC01 is in blue, MC07 in pink, MC11 in yellow and MC13 in green.

cT_FH subset activation state determined by PD1, ICOS, CCR7, and CD127.
Box-plots showing (a) PD1, (b) ICOS, (c) CCR7, and (d) CD127 expression across all cT_FH-like meta-clusters from children (n=13) with no stimulation (blue), and after in vitro stimulation with PfGARP (green) or PfSEA-1A (pink). Fifty percent of the data points are within the box limits, the solid line indicates the median, the dash line the mean and the whiskers indicate the range of the remaining data with outliers being outside that range.

Differences in abundance of antigen-specific cT_FH meta-clusters for adults and children.
(a) UMAP plots showing the 12 different cT_FH meta-clusters in adults (top three plots, n=13) and in children (bottom three plots, n=13) in the absence of stimulation or after in vitro stimulation with PfSEA-1A or PfGARP, from left to right, respectively. Colored circles highlight the meta-clusters showing differences in their abundance between adults and children for each condition. An EdgeR statistical plot was performed to assess the change in abundance of the 12 meta-clusters between adults and children after (b) no stimulation, (c) PfSEA-1A and (d) PfGARP stimulations. EdgeR plots indicate which meta-clusters are significantly abundant between two groups by using green color dots. The Y-axis is the - log10(p-value) and the X-axis is the log(FC). Green dots were statistically significant (p<0.05). Numbers next to the dots indicate the meta-cluster. (e) An abundance heatmap indicates the percentage (black numbers) of each meta-cluster within the total number of CD3^posCD4^posCXCR5^posCD25^neg cells for adults and children (concatenated from 13 participants in each group) under the different conditions: no stimulation (Neg) or stimulation with PfSEA-1A or PfGARP. The color scale ranges from high expression (red) to low/no expression (blue). The star in the heatmap indicates which meta-cluster is significantly abundant in children or adults based on the EdgeR results.

Diverse Pf-malaria antigen-specific expression patterns of cT_FH defining cytokines and transcription factors for children.
Heatmaps of Wilcoxon-paired two-tailed t-test p-values are shown for cT_FH meta-clusters comparing (a) PfSEA-1A and (b) Pf-GARP versus unstimulated PBMCs from children (n=13) for each cytokine (IFNγ, IL4, and IL21) and transcription factor (Bcl6 and cMAF). The color-scale indicates the significance of the p-value: white (non-significant, p>0.05), yellow (0.05>p>0.02), orange (0.02>p>0.005), and red (highly significant, p<0.005). The down-directed arrow indicates a decrease of expression from unstimulated to stimulated condition whereas no arrow indicates an increase of expression from unstimulated to stimulated condition. The cT_FH meta-clusters that co-expressed transcription factors were grouped as follows: Group 1 (blue line), Group 2 (green line), and Group 3 (purple line). Bar plots indicating mean with SD of the median intensity fluorescence (MFI) of (c) IFNγ, (d) Bcl6, and (e) cMAF for the cT_FH meta-clusters showing significant statistical differences between PfSEA-1A and PfGARP stimulations. p-values from Wilcoxon-paired two-tailed t-tests are indicated.

Limited Pf-malaria antigen-specific expression patterns of cT_FH defining cytokines and transcription factors for adults.
Heatmaps of Wilcoxon-paired two-tailed t-test p-values are shown for cT_FH meta-clusters comparing (a) PfSEA-1A or (b) Pf-GARP versus unstimulated PBMCs from adults (n=13) for each cytokine (IFNγ, IL4, and IL21) and transcription factors (Bcl6 and cMAF). The color-scale indicates the significance of the p-value: white (non-significant, p>0.05), yellow (0.05>p>0.02), orange (0.02>p>0.005) and red (highly significant, p<0.005). The down-directed arrow indicates a decrease of expression from unstimulated to stimulated condition whereas no arrow indicates an increase of expression from unstimulated to stimulated condition. The only cT_FH meta-clusters that expressed transcription factors were in Group 3 (purple box). Bar plots indicating mean with SD of the median intensity fluorescence (MFI) of (c) Bcl6, and (d) cMAF for the cT_FH meta-clusters showing significant statistical differences between PfSEA-1A and PfGARP stimulations. p-values from Wilcoxon-paired two-tailed t-tests are indicated.

Abundance of cT_FH meta-clusters stratified by anti-PfGARP IgG antibody levels.
UMAP and EdgeR analysis of (a) children (n=13) and (b) adults (n=13) where significant differences (p<0.05) in the abundance of the cT_FH meta-clusters are circled on high versus low PfGARP antibody level (left to right) UMAP and green dots on the volcano plot (far right).

Summary.
Illustration of combined findings showing remarkable differences in cT_FH subset abundance and Pf-malaria antigen-specific cytokine and transcription factor responses between children and adults residing in a malaria holoendemic region of Kenya. Made with BioRender.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Representative cytoplots of the extracellular flow staining.
Panel a shows the CXCR5 vs CD25 cytoplots on unfiltered, CD19^pos, CD3^pos, CD4^pos and CD8^pos cells from left to right. Panel b shows CD45RA vs CCR7 cytoplots on CD3^pos, CD8^pos, CD4^pos and CD4^posCXCR5^pos cells from left to right. Panel c shows CCR6 vs CXCR3 staining on CD4^posCXCR5^pos cells. Panel d shows CD127 vs CD40L expression within CD4^posCXCR5^pos cells by unstimulated and SEB stimulated cells. Panel e shows ICOS vs PD1 cytoplots after no stimulation or SEB 6h stimulation.

EdgeR analysis of the CD4^pos, CD4^posCXCR5^pos, CD8^pos and CD14/CD19^pos populations under different stimulation conditions in adults (n=15) and children (n=14).
Volcano plot from EdgeR analysis comparing CD4^pos, CD4^posCXCR5^pos, CD8^pos, and CD14/CD19^pos populations from adult PBMCs under the following conditions: (a) unstimulated versus PfSEA-1A, (b) unstimulated versus PfGARP, and (c) PfSEA-1A versus PfGARP. Volcano plot from EdgeR analysis comparing CD4^pos, CD4^posCXCR5^pos, CD8^pos,and CD14/CD19^pos populations from children PBMCs under the following conditions: (d) unstimulated versus PfSEA-1A, (e) unstimulated versus PfGARP, and (f) PfSEA-1A versus PfGARP. The green dots are statistically significant whereas the black dots are not, here no green dots were observed.

Characterization of T_FH subsets using CXCR5, CXCR3, CCR6, and CD45RA expression.
Bar plots showing (a) CXCR5, (b) CD45RA, (c) CCR6, and (d) CXCR3 expression across all cT_FH-like meta-clusters from adults (n=13) under no stimulation (blue), PfGARP (green) and PfSEA-1A (pink) stimulations. Fifty percent of the data are within the box limits, the solid line indicates the median, the dash line the mean and the whiskers indicate the range of the remaining data with outliers being outside that range.

CCR6 versus CXCR3 representative cytoplots. a.
CCR6 vs CXCR3 expression from activated cT_FH2-like MC01 (in blue), quiescent cT_FH1-like MC07 (in pink), activated cT_FH1/17-like MC11 (in orange) and activated cT_FH17-like MC13 (in green). Undetermined meta-clusters was superposed to the sus-mentioned metacluster: activated cT_FH1or2-like MC03 (in black) (b); activated cT_FH1or2-like MC04 (in black) (c); quiescent cT_FH1or2-like MC05 (in black) (d); quiescent cT_FH1or2-like MC08 (in black) (e).

Cartoon of T_FH subsets and their state of activation.

State of activation of cT_FH subsets from adults determined by PD1, ICOS, CCR7, and CD127.
Bar plots showing (a) PD1, (b) ICOS, (c) CCR7 and (d) CD127 expression across all cT_FH-like meta-clusters in adults (n=13) under no stimulation (blue), PfGARP (green) and PfSEA-1A (pink) stimulations. Fifty percent of the data are within the box limits, the solid line indicates the median, the dash line the mean and the whiskers indicate the range of the remaining data with outliers being outside that range.

CD40L expression across stimulation and meta-clusters.
Dot plots of CD40L expression after manual gating based on unstimulated condition from children (n=13) (a) and adults (n=13) (b) cT_FH meta-clusters.

Abundance of the cT_FH meta-clusters within children with low or high levels of HRP2 antibodies.
(a) UMAP plot showing the clustering of the 13 different cT_FH meta-clusters (MC15) in children with low levels of HRP2 (on the left, n=6) and high levels of HRP2 (on the right, n=6) in the absence of stimulation. Each color represents a meta-cluster. The red circles highlight the meta-cluster showing differences in its abundance between the two groups of children. (b) An EdgeR statistical plot was performed to assess the abundance of the 13 meta-clusters between unstimulated PBMCs from both groups of children; the Y-axis being -log10(p-value) and the X-axis shows the log(FC). The green dot (MC04) is statistically significant whereas the black dots are not. (c) An abundance heatmap showing the distribution (in %) of each meta-cluster within the two groups of children (both concatenated unstimulated PBMCs from six participants). The color scale ranges from high abundance (red) to low abundance (blue).

Clustered heatmap of the cytokines and transcription factors expressed from cT_FH meta-clusters in adults and children under the different stimulation conditions.
Heatmap of Bcl6, IFNγ, IL21, IL4, and cMAF expression from the 13 cT_FH meta-clusters from concatenated data from (a) children (n=13) and (b) adults (n=13), after stimulation by PfSEA-1A or PfGARP, or without any stimulation as indicated in the name of each row. The color scale ranges from high expression (red) to low/no expression (blue).

Bar plots of cytokines expressed under the different conditions of stimulation in children (n=13).
Comparison of mean fluorescent intensity (MFI) of (a) IFNγ, (b) IL4 cytokines expression after PfSEA-1A (pink) or PfGARP (green) stimulation or no stimulation control (blue). Bar plots indicate mean with SD. Wilcoxon-paired two-tailed t-tests were performed and p-values are indicated.

Bar plots of transcription factors expressed under the different conditions of stimulation in children (n=13).
Comparison of mean fluorescent intensity (MFI) of (a) Bcl6 and (b) cMAF expression after PfSEA-1A (pink) or PfGARP (green) stimulation or no stimulation control (blue). Bar plots indicate mean with SD. Wilcoxon-paired two-tailed t-tests were performed and p-values are indicated.

Bar plots of cytokines expressed under the different conditions of stimulation in adults (n=13).
Comparison of mean fluorescent intensity (MFI) of (a) IFNγ, (b) IL4 and (c) IL21 and cytokines expression after PfSEA-1A (pink) or PfGARP (green) stimulation or no stimulation control (blue). Bar plots indicate mean with SD. Wilcoxon-paired two-tailed t-tests were performed and p-values are indicated.

Bar plots of transcription factors expressed under the different conditions of stimulation in adults (n=13).
Comparison of mean fluorescent intensity (MFI) of (a) Bcl6 and (b) cMAF expression after PfSEA-1A (pink) or PfGARP (green) stimulation or no stimulation control (blue). Bar plots indicate mean with SD. Wilcoxon-paired two-tailed t-tests were performed and p-values are indicated.

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