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

Abstract

Circulating T-follicular helper (cT_FH) cells have the potential to provide an additional correlate of protection against Plasmodium falciparum (Pf) as they are essential to promote B-cell production of long-lasting antibodies. Assessing the specificity of cT_FH subsets to individual malaria antigens is vital to understanding the variation observed in antibody responses and identifying promising malaria vaccine candidates. Using spectral flow cytometry and unbiased clustering analysis, we assessed antigen-specific cT_FH cell recall responses in vitro to malaria vaccine candidates Pf-schizont egress antigen-1 (PfSEA-1A) and Pf-glutamic acid-rich protein (PfGARP) within a cross-section of children and adults living in a malaria-holoendemic region of western Kenya. In children, a broad array of cT_FH subsets (defined by cytokine and transcription factor expression) were reactive to both malaria antigens, PfSEA-1A and PfGARP, while adults had a narrow profile centering on cT_FH17- and cT_FH1/17-like subsets following stimulation with PfGARP only. Because T_FH17 cells are involved in the maintenance of memory antibody responses within the context of parasitic infections, our results suggest that PfGARP might generate longer-lived antibody responses compared to PfSEA-1A. These findings have intriguing implications for evaluating malaria vaccine candidates as they highlight the importance of including cT_FH profiles when assessing interdependent correlates of protective immunity.

Keywords: Kenya; P. falciparum; T-follicular helper; immunology; inflammation; pf-malaria; vaccine candidates.

Figure 1.. 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 standard deviation (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 median fluorescence intensity (MFI) values are the antigen-specific MFI values minus the BSA background. Mann-Whitney tests were performed.

Figure 2.. 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 five 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. Uniform Manifold Approximation and Projection (UMAP) plots showing five 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 peripheral blood mononuclear cells (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.

Figure 2—figure supplement 1.. 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 6 hr stimulation.

Figure 2—figure supplement 2.. 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 peripheral blood mononuclear cells (PBMCs) under the following conditions: (a) unstimulated vs PfSEA-1A, (b) unstimulated vs PfGARP, and (c) PfSEA-1A vs 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 vs PfSEA-1A, (e) unstimulated vs PfGARP, and (f) PfSEA-1A vs PfGARP. The green dots are statistically significant, whereas the black dots are not; here, no green dots were observed.

Figure 3.. 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 five CD4^posCXCR5^posCD25^neg nodes as 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.

Figure 3—figure supplement 1.. 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 dashed line the mean, and the whiskers indicate the range of the remaining data with outliers being outside that range.

Figure 4.. 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.

Figure 4—figure supplement 1.. CCR6 vs 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 were 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).

Figure 4—figure supplement 2.. 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 dashed line the mean, and the whiskers indicate the range of the remaining data with outliers being outside that range.

Figure 4—figure supplement 3.. 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.

Figure 5.. Differences in abundance of antigen-specific cT_FH meta-clusters for adults and children.

(a) Uniform Manifold Approximation and Projection (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.

Figure 5—figure supplement 1.. Abundance of the cT_FH meta-clusters within children with low or high levels of HRP2 antibodies.

(a) Uniform Manifold Approximation and Projection (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 peripheral blood mononuclear cells (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).

Figure 6.. 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 vs unstimulated peripheral blood mononuclear cells (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 (nonsignificant, 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 standard deviation (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.

Figure 6—figure supplement 1.. 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).

Figure 6—figure supplement 2.. 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 standard deviation (SD). Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated.

Figure 6—figure supplement 3.. 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 standard deviation (SD). Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated.

Figure 6—figure supplement 4.. Manually gated transcription factors expression upon stimulation.

(a) Bcl6 vs cMAF cytoplots from the six runs overlaid FMOs. (b) Percentage of CD4^posCXCR5^posBcl6^pos cells across stimulation in children (left) and adults (right). Bar plots indicate mean with standard deviation (SD). Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated. (c) Percentage of CD4^posCXCR5^poscMAF^pos cells across stimulation in children (left) and adults (right). Bar plots indicate mean with SD. Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated. Manually gated positive Bcl6 cells overlaid on the Uniform Manifold Approximation and Projection (UMAP) of the CD4^posCXCR5^posCD25^neg cells across stimulation in children (d) and adults (e). (f) Contour plots with outliers of cMAF vs Bcl6 expression upon stimulation for each meta-cluster.

Figure 6—figure supplement 5.. Manually gated IFNγ and IL21 expression upon stimulation.

(A) Cytoplot of IFNγ vs CXCR5 expression from CD4 T cells upon stimulation. (B) Percentage of CD4^posIFNγ^pos cells across stimulation. (C) Percentage of CD4^posCXCR5^posIFNγ^pos cells across stimulation. (D) Spaghetti plot of the CD4^posCXCR5^posIFNγ^pos cells across stimulation, each color represents a participant. (E) Cytoplots of IFNγ vs FSC-A expression from CD89 T cells upon stimulation. (F) Percentage of CD8^posIFNγ^pos cells across stimulation. Manually gated positive IFNγ cells overlaid on the Uniform Manifold Approximation and Projection (UMAP) of the CD4^posCXCR5^posCD25^neg cells across stimulation in children (G) and adults (H). (I) Cytoplots of IFNγ vs IL21 expression from six overlaid FMOs and CD4^posCXCR5^pos T cells upon stimulation. (J) Percentage of CD4^posIL21^pos cells across stimulation in children. (K) Percentage of CD4^posIL21^pos cells across stimulation in adults. Manually gated positive IL21 cells overlaid on the UMAP of the CD4^posCXCR5^posCD25^neg cells across stimulation in children (L) and adults (M). (N) Contour plots with outliers of IFNγ vs IL21 expression upon stimulation for each meta-cluster. Bar plots indicate mean with standard deviation (SD). Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated.

Figure 6—figure supplement 6.. Manually gated IL4 and IL21 expression upon stimulation.

(A) Cytoplot of IL4 vs IL21 expression from six overlaid FMO and CD4^posCXCR5^pos T cells upon stimulation. Bar plots indicate mean with standard deviation (SD). Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated. Percentage of CD4^posCXCR5^posIL4^pos cells across stimulation in children (B) and adults (C). Manually gated positive IL4 cells overlaid on the Uniform Manifold Approximation and Projection (UMAP) of the CD4^posCXCR5^posCD25^neg cells across stimulation in children (D) and adults (E). IL4 mean fluorescence intensity (MFI) for all meta-clusters represented in bar plots across stimulation in children (F) and adults (G), negative control in blue, PfSEA-1A in pink, and PfGARP in green. Wilcoxon paired two-tailed t-tests were performed, and significant p-values are indicated with * equal p<0.05. (H) Contour plots with outliers of L4 vs IL21 expression upon stimulation for each meta-cluster.

Figure 7.. 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 vs unstimulated peripheral blood mononuclear cells (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 (nonsignificant, 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 standard deviation (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.

Figure 7—figure supplement 1.. 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 standard deviation (SD). Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated.

Figure 7—figure supplement 2.. 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 standard deviation (SD). Wilcoxon paired two-tailed t-tests were performed, and p-values are indicated.

Figure 8.. Abundance of cT_FH meta-clusters stratified by anti-PfGARP IgG antibody levels.

Uniform Manifold Approximation and Projection (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 vs low PfGARP antibody level (left to right) in the UMAP plot and green dots on the volcano plot (far right).

Figure 9.. 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.

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