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, United States
Department of Pathology and Laboratory Medicine, Brown University, Providence, United States
Center for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya

https://doi.org/10.7554/eLife.98462.2

Open access
Copyright information

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 the mean with SD. (b) IgG antibody levels against PfSEA-1A and PfGARP comparing children (n=15) and adults (n=15). The bar plots indicate the mean with SD. Net MFI values are the antigen-specific MFI values minus the BSA background. Mann-Whitney tests were performed.

Cluster visualization of immune cell types and their abundance.
(a) A 100- node and 75-meta-cluster 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 were used for the downstream analysis. The colored scale was based on the median arcsinh-transformed marker expression. UMAP plots showing 5 clusters defined as follows: CD14^pos and CD19^pos (orange), CD4^pos (blue), CD4^posCXCR5^pos (red), CD8^pos (purple), and CD4^posCD25^pos (green) from PBMCs isolated from (b) adults (n=15) and (c) children (n=14) that were unstimulated, stimulated with PfSEA-1A, or stimulated with PfGARP, respectively, from left to right.

Characterization of T_FH subsets by CXCR5, CXCR3, CCR6, and CD45RA expression.
(a) A new 25-node and 15 meta-cluster 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 as 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 dashed line indicates 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 blue, MC07 is pink, MC11 is yellow, and MC13 is 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 dashed line indicates 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 or stimulation with (c) PfSEA-1A or (d) PfGARP. 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, IL-4, and IL-21) 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 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. The 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, IL-4, and IL-21) 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 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 indicate the mean with SD of the median fluorescence intensity (MFI) of (c) Bcl6 and (d) cMAF for the cT_FH meta-clusters showing significant statistical differences between PfSEA-1A and PfGARP stimulations. The 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 analyses 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) in the UMAP plot 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. Figure created 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.

Sign up for email alerts