CXCR5+PD-1++ CD4+ T cells colonize infant intestines early in life and promote B cell maturation

Abstract

Gastrointestinal infections are a major cause for serious clinical complications in infants. The induction of antibody responses by B cells is critical for protective immunity against infections and requires CXCR5⁺PD-1⁺⁺ CD4⁺ T cells (T_FH cells). We investigated the ontogeny of CXCR5⁺PD-1⁺⁺ CD4⁺ T cells in human intestines. While CXCR5⁺PD-1⁺⁺ CD4⁺ T cells were absent in fetal intestines, CXCR5⁺PD-1⁺⁺ CD4⁺ T cells increased after birth and were abundant in infant intestines, resulting in significant higher numbers compared to adults. These findings were supported by scRNAseq analyses, showing increased frequencies of CD4⁺ T cells with a T_FH gene signature in infant intestines compared to blood. Co-cultures of autologous infant intestinal CXCR5⁺PD-1^+/-CD4⁺ T cells with B cells further demonstrated that infant intestinal T_FH cells were able to effectively promote class switching and antibody production by B cells. Taken together, we demonstrate that functional T_FH cells are numerous in infant intestines, making them a promising target for oral pediatric vaccine strategies.

Keywords: Antibodies; B cells; Intestine; Pediatrics; TFH cells.

Fig. 1

Increased numbers of CXCR5⁺PD-1⁺⁺ CD4⁺ T populate infant intestines and decline with age. A Representative flow cytometric plots of the gating strategies used to identify CXCR5⁺PD-1⁺⁺ CD4⁺T cells in intraepithelial lymphocytes (IEL) and lamina propia lymphocytes (LPL) in infant (dark blue) and adult intestines (light blue). B Frequencies (%) and cell counts/cm² of epithelial and lamina propria-derived CXCR5⁺ CD4⁺ T cells in infant and adult intestines. C Frequencies (%) and cell counts/cm² of epithelial and lamina propria-derived CXCR5⁺PD-1⁺⁺ CD4⁺ T cells in infant and adult intestines. D Representative flow cytometric plot of fetal intestinal CXCR5⁺PD-1⁺⁺ CD4⁺ T cells and frequencies (%) of lamina propria-derived CXCR5⁺PD-1⁺⁺ CD4⁺ T cells in fetal compared to infant and adult samples. Medians and interquartile ranges (IQRs) are depicted in all figures. (Infant IEL % n = 7; adult IEL % n = 10; infant IEL cell counts/cm² n = 5; adult IEL cell counts/cm² n = 8; infant LPL % n = 11; adult LPL % n = 11; infant LPL cell counts/cm² n = 7; adult LPL cell counts/cm² n = 10; fetal LPL % n = 3). Mann–Whitney test was used for comparisons. Only significant p values are shown

Fig. 2

Infant intestinal CXCR5⁺PD-1⁺⁺ CD4⁺ T cells exhibit a tissue-resident memory phenotype. A Representative flow cytometric plots to define i) left: memory subsets based on CCR7 and CD45RA expression in CXCR5⁺PD-1⁺⁺ CD4⁺ T cells and ii) right: CD103 and CD69 expression on CXCR5⁺PD-1⁺⁺ CD4⁺ T cells. B Frequencies (%) of naive (CCR7⁺CD45RA⁺), central memory (CCR7⁺CD45RA−) and effector memory (CCR7^-CD45RA⁻) T cell populations in CXCR5⁺PD-1⁺⁺CD4⁺ T cells comparing infant and adult intestines. C Frequencies (%) of CD69⁺ cells and CD69⁺CD103⁺ cells within CXCR5⁺PD-1⁺⁺ CD4⁺ T cells comparing infant and adult intestines. Medians and IQRs are depicted in all figures. (For B and C left: infant n = 9; adult n = 8. For C right: infant n = 6; adult n = 4). Mann–Whitney test was used to compare infant and adult samples. Only significant p values are shown

Fig. 3

Infant intestinal CXCR5⁺PD-1⁺⁺ CD4⁺ T cells have a T_FH phenotype. A Representative flow cytometric plots of the expression levels of Bcl-6 in CXCR5⁺PD-1⁺⁺ CD4⁺ T cells and CXCR5⁻PD−1⁻CD4⁺ T cells in infant and adult intestines. B Frequencies (%) of Bcl-6 expressing intestinal CXCR5⁺PD-1⁺⁺ cells (green) and CXCR5⁻PD-1⁻ CD4⁺ T cells (orange) in infants and adults. (Infant CXCR5⁺PD-1⁺⁺ % n = 8; infant CXCR5⁻PD-1⁻ % n = 10; adult CXCR5⁺PD-1⁺⁺ % n = 7; adult CXCR5⁻PD-1⁻ % n = 10). Medians and IQRs are depicted in all figures. Mann–Whitney test was used to compare infant and adult samples, and Wilcoxon matched-pairs signed ranked test was used to compare CXCR5⁺PD-1⁺⁺ and CXCR5⁻PD-1⁻ CD4⁺ T cells. Only significant p values are shown

Fig. 4

Infant and adult intestinal T_FH cells share a similar transcriptomic profile. A UMAP plot of CD4⁺ T cells analyzed by scRNAseq from infant intestinal (n = 3 donors), adult intestinal (n = 3 donors), infant blood (n = 2 donors) and adult blood (n = 3 donors) samples. In total, 2305 cells were analyzed. B Identification of 12 clusters of T helper subtypes using a shared nearest neighbor (SNN) modularity optimization-based clustering algorithm. C Enrichment of published T_FH signature [28, 29] (based on sorted CXCR5⁺CD4⁺ T cells) projected on the UMAP plot. D Quantification of the T_FH signature in single cells, separately for each cluster from B. Cluster 7 stands out as the cluster with the most prominent enrichment of the T_FH signature. E Principal component analysis (PCA) of the transcriptome of T_FH cells (cluster 7) aggregated for each donor. F Differentially expressed genes between infant and adult intestinal T_FH cells (cluster 7), showing only four genes significantly differentially regulated after multi comparison correction (statistical testing using DEseq2). Comparisons between the remaining sample types are shown in Fig. S3G–I. G Expression of significantly differentially expressed genes from F, based on aggregation of single cells in cluster 7 for each donor. Box plots in D and G centered on median, bounds defined between the 25th and 75th percentile with minimum and maximum defined as median ± 1.5 × interquartile range and whiskers extending to the lowest/highest value within this range

Fig. 5

Infant intestinal CD4⁺ T cells produce IL-21 and TNF. A Representative flow cytometric plots of viable infant and adult intestinal CD4⁺ T cells (CD4⁺ CD3⁺, unstimulated;   CD3⁺CD8^-, PMA/ionomycin-stimulated) lamina propria-derived lymphocytes showing IL-21 and TNF expression. B Frequencies (%) of infant and adult IL-21⁺ CD4⁺ T cells in unstimulated (−) and stimulated (p/i) conditions. C Frequencies (%) of infant and adult TNF⁺ CD4⁺ T cells in unstimulated (-) and stimulated (p/i) conditions. D Representative flow cytometry plots showing ICOS expression and CD40L expression on CXCR5⁺PD-1⁺⁺ CD4⁺ T cells (green) and CXCR5⁻PD-1⁻ CD4⁺ T cells (orange) in infant and adult intestines. E ICOS expression of CXCR5⁺PD-1⁺⁺ CD4⁺ T cells relative to the sample corresponding CXCR5⁻PD-1⁻ CD4⁺ T cells. F CD40L expression of CXCR5⁺PD-1⁺⁺ CD4⁺ T cells relative to the sample corresponding CXCR5⁻PD-1⁻ CD4⁺ T cells. Medians and IQRs are depicted in all figures. (For B infant n = 5; adult n = 5. For C infant n = 11; adult n = 7. For E infant n = 6; adult n = 5. For F infant n = 6; adult n = 6). Wilcoxon matched-pairs signed rank test was used to compare unstimulated/stimulated samples, Mann-Whitney test was used to compare cytokine productions by infant and adult cells; and Wilcoxon signed rank test was used to compare ICOS and CD40L expression by CXCR5⁺PD⁻1⁺⁺ and CXCR5⁻PD-1⁻ CD4⁺ T cells. Only significant p values are shown

Fig. 6

Memory B cells in infant intestines. A Representative flow cytometric plots of the gating strategies showing B cell maturation in infants (upper plots) and adults (lower plots). SM switched memory, NSM non-switched memory, DNM double negative memory. B Frequencies (%) of CD19⁺ of total CD45⁺ cells in infants and adults. C Frequencies (%) of GC (germinal center; Bcl-6⁺Ki67⁺) and CD138⁺ cells of total CD19⁺ cells in infants and adults. D Frequencies (%) of naive and SM cells of total CD20⁺ cells in infants and adults. E Frequencies (%) of CD24⁺ and IgM^- of total SM cells in infants and adults. F Frequencies (%) of IgA⁺ and IgG⁺ of total IgM^- cells in infants and adults. G Representative immunohistochemistry (IHC) images of tissue samples from a newborn infant (upper panel), a 3 month-old (middle panel) and 18 month-old infant (lower panel) showing the expression levels of the transcription factor Bcl-6, CD20 and IgA. Images are representative of the analyses of 13 intestinal samples. Scale bars indicate 0.1 mm. Medians and IQRs are depicted in all figures. (For B, C, D, E, F infant n = 4; adult n = 4.). Mann–Whitney test was used for comparisons. Only significant p values are shown

Fig. 7

Infant intestinal CXCR5⁺PD-1^+/− CD4⁺ T cells promote infant intestinal B cell maturation. A Representative flow cytometric plots of B cell analyses of B cell cultures alone or upon co-culture with CXCR5⁺PD-1^+/− CD4⁺ T cells (lower plots), showing IgG, IgA and Bcl-6 expressions. B Frequencies (%) of IgG⁺ (top left), IgA⁺ (top right) and Bcl-6⁺ (bottom) in B cells cultured alone and in co-culture with autologous CXCR5⁺PD-1^+/− cells. C Levels of IgG (left) and IgA (right) present in culture supernatants of B cells cultured alone and in co-culture with CXCR5⁺PD-1^+/− cells measured by ELISA. Limit of detection for each ELISA experiment is depicted with a horizontal line in the plot. Medians and IQRs are depicted in all figures. N = 5 intestinal samples. For 3 of the donors, 3 replicates were performed. Each dot represents one donor/replicate. Wilcoxon matched-pairs signed rank test was used for comparisons. Only significant p values are shown