Influenza vaccination stimulates maturation of the human T follicular helper cell response

Abstract

The differentiation and specificity of human CD4⁺ T follicular helper cells (T_FH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4⁺ T_FH cell response. The first vaccination induced an increase in the frequency of circulating T_FH (cT_FH) and LN T_FH cells at week 1 postvaccination. This increase was transient for cT_FH cells, whereas the LN T_FH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of T_FH cells in the LN, including pre-T_FH cells, memory T_FH cells, germinal center (GC) T_FH cells and interleukin-10⁺ T_FH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC T_FH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific T_FH cell clonal lineages, including multiple responses targeting internal influenza virus proteins, and found that each T_FH cell state was attainable within a clonal lineage. Thus, human T_FH cells form a durable and dynamic multitissue network.

Fig. 1. Influenza vaccination stimulates a CD4⁺ T_FH cell response in blood and LN of humans.

a, Schematic representation indicating the time points of PBMC (D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1 and D120_Y1) and LN (D0_Y1, D5_Y1, D12_Y1, D26_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2) samples in five donors (321-04, 321-05, 321-07, 321-08 and 321-11) postvaccination with Flucelvax QIV on D0_Y1 (2018–2019 season) and D0_Y2 (2019–2020 season) 1 year apart. b, Representative flow cytometry plots of circulating CXCR5⁺CD38⁺ T_FH cells (cT_FH cells) in PBMCs from donor 321-05 at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1 and D180_Y1, gated on CD45⁺CD14⁻CD19⁻CD3⁺CD4⁺ cells. c, Frequency of ICOS⁺CD71⁺ cT_FH cells in PBMCs from donor 321-05 at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1 and D180_Y1, gated on CXCR5⁺CD38⁺ T_FH cells as in b. d, Representative flow cytometry plots of CXCR5⁺PD-1⁺ LN T_FH cells in donor 321-05 at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1 and D180_Y1, gated on CD14⁻CD19⁻CD4⁺ T cells. e, Frequency of CD27⁺BCL6⁺ LN T_FH cells in 321-05 at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1 and D180_Y1, gated on CXCR5⁺PD-1⁺ T_FH cells as in d. f, Frequency of cT_FH, LN T_FH and CD19⁺IgD^loCD20^hiCD38^int LN GC B cells in 321-05 at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1 and D180_Y1. Shown as a percent of total CD4⁺ T cells or CD19⁺ B cells on log₁₀ scale.

Fig. 2. scGEX and TCR profiling capture diverse T cell phenotypes in blood and LN after influenza vaccination.

a, UMAP projection of the total T cell dataset generated from the PBMC (at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1 and D120_Y1) and LN (at D0_Y1, D5_Y1, D12_Y1, D26_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2) samples from donors 321-04, 321-05, 321-07 and 321-08 annotated by GEX cluster (n = 154,109 cells with 127,329 unique TCR clonotypes). b, UMAP projection of the total T cell dataset annotated by T cell phenotype as CCR7^hiSELL^hi T_N, CCR7^loSELL^loKLRG1⁺ T_E cells or T_M cells or PDCD1⁺CXCR5⁺ T_FH or FOXP3⁺ T_reg cells. c, Feature plots displaying distinguishing T cell markers in the total T cell dataset.

Fig. 3. Influenza vaccination stimulates the differentiation of distinct T_FH cell subsets.

a–d, UMAP projections of the T_FH/T_reg subset annotated by scGEX cluster (a), donor (b), tissue (c) and time postimmunization (d) in a subset of the total T cell dataset in Fig. 2 that contained all cells clonally related to those in the T_FH/T_reg cell cluster C14 from the PBMC and LN of all donors (n = 6,109 cells with 4,418 unique TCR clonotypes). e, UMAP projection of the T_FH/T_reg subset annotated as CCR7^loCXCR5^loPDCD1^loICOS⁺ pre-T_FH/mem-T_FH cells, CCR7⁻CXCR5^hiPDCD1^hiICOS⁺ GC T_FH cells, CCR7⁻CXCR5^hiPDCD1^hiICOS⁺LAG3⁺ IL-10⁺ T_FH cells, FOXP3⁺ T_reg cells, MKI67⁺ cycling cells or CCR7⁺CD4⁺ T_N cells. f, Dot plot of select T_FH cell and T_reg cell subset-specific markers in T_FH/T_reg subset grouped by each subset (excluding cycling) defined in e. g,h, Reclustering and feature plots of CCR7^loCXCR5^loPDCD1^loICOS⁺ pre-T_FH/mem-T_FH cells (g) and CCR7⁻CXCR5^hiPDCD1^hiICOS⁺ GC T_FH cells (h) displaying marker genes associated with scGEX clusters within the pre-T_FH/mem-T_FH and GC T_FH cells.

Fig. 4. Influenza vaccination alters T_FH cell metabolism and signaling with time.

a, Enrichr analysis showing enriched GO biological processes in using LN pre-T_FH/mem-T_FH, IL-10⁺ T_FH, GC T_FH and T_reg cells from the T_FH/T_reg subset from donors 321-04 and 321-05 at time points D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2. Marker DEGs for the four subsets were redefined by comparing them against the global background of subsetted LN pre-T_FH/mem-T_FH, GC T_FH, IL-10⁺ T_FH and T_reg cells included in the analysis. b, GSVA across D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D180_Y1, D0_Y2, D7_Y2, D28_Y2, D60_Y2 and D120_Y2 in LN T_FH cells from donor 321-05 (excluding T_reg cells) for top T_FH cell-specific GO terms for a.

Fig. 5. Influenza vaccination elicits dynamic alterations in T_FH cell composition.

a, Relative frequency of LN GC T_FH cell, GC B cells, IL-10⁺ T_FH cells, pre-T_FH/mem-T_FH cell and T_reg cell subsets in donor 321-05 at time points D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D180_Y1, D0_Y2, D7_Y2, D28_Y2, D60_Y2 and D120_Y2. b–d, UMAP projections of LN pre-T_FH/mem-T_FH, GC T_FH, IL-10⁺ T_FH and CD4⁺ T_N cells from all donors at D0_Y1, D5_Y1, D12_Y1, D26_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2 subsetted from the T_FH/T_reg subset, reclustered and subjected to pseudotime analysis showing calculated pseudotime values with selected root nodes where pseudotime scale was set to zero are indicated by black points on the left side of the projection (b), time points (c) or T_FH cell subset (d). n = 4,418 cells. e, Density plots of pseudotime value distributions by T_FH cell subset in the division of the T_FH/T_reg subset defined in b. f, Scatter plot of log₁₀ normalized expression of indicated T_FH cell subset marker genes versus pseudotime values per cell in the division of the T_FH/T_reg subset defined in b. Each dot represents a cell, and the color indicates its assigned T_FH cell subset.

Fig. 6. Influenza vaccination leads to T_FH cell clonal expansion.

a, Relative frequency of all subsets of T_FH cells in LN (T_FH/T_reg subset excluding T_reg and T_N cells) from donors 321-04 and 321-05 over D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2 time points. b, TCR clonality of all subsets of T_FH cells in LN (T_FH/T_reg subset excluding T_reg and T_N cells) from donors 321-04 and 321-05 over D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2 time points by inverse D50 index. c, Scatter plot comparing the relative frequency and clonality from a and b (n = 20). Correlation analyses were performed using the Pearson correlation metric, and P values were computed using two-sided t test. d, Network graph depicting the connection between T_FH cell clonal lineages in PBMCs and LN in donor 321-05 at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D180_Y1, D0_Y2, D7_Y2, D28_Y2, D60_Y2 and D120_Y2 time points. Node sizes correspond to the number of unique clones, and edge widths correspond to the number of clones connecting each node.

Fig. 7. Vaccination evokes maturation of influenza-specific T_FH cell clonal lineages.

a, Number of cells detected in PBMCs (top) and LN (bottom) for T_FH cell clonal lineages T1–T12 in donor 321-05 at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D180_Y1, D0_Y2, D7_Y2, D28_Y2, D60_Y2 and D120_Y2. b, Relative abundance of T_FH cell clonal lineages T1–T12 in bulk TCRβ sequencing of sorted cT_FH cells and LN T_FH cells from donor 321-05 at D5_Y1, D12_Y1, D28_Y1 and D60_Y1. c, Frequency of CD69⁺ cells in T1, T3 and T12 cell lines after 24 h of coculture with aAPCs expressing HLA-DR3*03:01, HLA-DPA1*01:03/DPB1*13:01 or HLA-DR5*01:01 pulsed with peptides derived from influenza M2, M1, NA or NP, and in the T6 cell line after 24 h of coculture with B cells expressing HLA-DPA1*01:03/DPB1*04:02 pulsed with NA peptides. DMSO alone was used as a control (n = 2–4 technical replicates per condition). Representative of two experiments. d, Frequency of IFNγ⁺ T11 Jurkat T cell line after 24 h coculture with aAPCs transfected with plasmids containing full HA protein, residues 1–460 (HA 460), 1–360 (HA 360), 1–260 (HA 260), 1–160 (HA 160) or the empty vector control (unstim; n = 2 technical replicates per condition). Representative of n = 2 experiments. e, Heatmap of pre-T_FH/mem-T_FH, GC T_FH and IL-10⁺ T_FH cells gene set module scores for T_FH cell clonal lineages T1–T12 from donor 321-05 across time points in a. f, UMAP projection showing the localization of all cells encompassed by T_FH cell clonal lineages T1–T12 assessed in donor 321-05 across time points from within the embedding from Fig. 2e. Source data

Extended Data Fig. 1. T_FH flow gating strategy and donor 321-04 LN T_FH and cT_FH frequencies.

a,b, Gating strategies for defining activated cT_FH cells (CXCR5⁺CD38⁺ICOS⁺CD71⁺; a) and LN GC T_FH cells (CXCR5⁺PD⁻1⁺BCL-6⁺; b). c, Frequency of CXCR5⁺CD38⁺ cT_FH in PBMCs for donor 321-04 in year 1 determined using flow cytometry. Shown as percent of total CD4⁺ T cells. d, Frequency of CXCR5⁺PD-1⁺BCL-6⁺ GC T_FH and IgD^loCD20^hiCD38^int GC B cell in LN biopsies taken from donor 321-04 in year 1 determined using flow cytometry. Shown as percent of total CD4⁺ T cells or total CD19⁺ B cells.

Extended Data Fig. 2. Single-cell gene expression and TCR profiling capture diverse T cell phenotypes in blood and LN after influenza vaccination.

a, Dot plot showing the expressions of marker genes for each gene expression cluster of the total T cell dataset generated from the PBMC (at D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1 and D120_Y1) and LN (at D0_Y1, D5_Y1, D12_Y1, D26_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2) samples from donors 321-04, 321-05, 321-07 and 321-08 annotated by GEX cluster. n = 154,109 cells with 127,329 unique TCR clonotypes. b, Frequency of cells by donor within each gene expression cluster. c, 2D UMAP projection of the total T cell dataset colored by log₁₀(1 + n) transformed clone sizes. d, Distribution of log₁₀(1 + n) transformed clone sizes across GEX clusters. e, Frequency of cells by donor within each gene expression cluster.

Extended Data Fig. 3. B cell subset frequency correlation and marker genes for T_FH/T_reg subset.

a, Heatmap of Pearson correlation between frequency of LN T_FH (all T_FH subsets) and naive, resting memory, activated, GC and plasmablast (PB) B cell subsets in the lymph node. b, Scatter plot comparing the relative frequencies of germinal center B cells versus T_FH cells in LN samples. Correlation analyses were performed using the Pearson correlation metric and P values were computed using two-sided t-tests. c, Heatmap of the top marker genes for each GEX cluster for the T_FH clonal lineages subset. d, Volcano plot of DEGs between indicated T_FH subset, T_reg cells and T_N cells. e, Violin plots of mouse full T_FH module score as defined in ref. in human T_FH cells from this study grouped by subset.

Extended Data Fig. 4. Influenza-vaccination stimulated alterations in TFH metabolism and signaling in donor 321-04.

GSVA of donor 321-05 LN T_FH cells with respect to time for T_FH-specific upregulated GO terms.

Extended Data Fig. 5. Influenza-vaccination induced changes to T_FH composition and phenotypes over time in donor 321-04.

a, Relative frequencies of pre/memory, GC and IL-10⁺ T_FH subsets in LN samples from donor 321-04 over time. b, Volcano plots showing DEGs within LN GC T_FH cells (all subsets) from donor 321-05 across matched time points (or all time points) after vaccination for years 1 and 2. D5_Y1 vs D7_Y2 (top), D28_Y1 vs D28_Y2 (middle) and year 1 vs year 2 (bottom). c, Heatmap of scaled GEX in LN GC T_FH cells from donor 321-05 at indicated time points. Genes shown are the top 10 marker genes for each time point from b. d, Violin plots showing expressions of IL21, CXCL13 and CXCR5 in LN GC T_FH cells for donors 321-04 (top) and 321-05 (bottom) at time points D0_Y1, D5_Y1, D12_Y1, D28_Y1, D60_Y1, D90_Y1, D120_Y1, D180_Y1, D0_Y2, D7_Y2, D14_Y2, D28_Y2, D60_Y2, D90_Y2 and D120_Y2. e, Comparison of somatic hypermutation frequency of QIV-specific antibody lineages to LN GC T_FH frequency after year 1 vaccination. The box-and-whisker plots show the distribution of SHM frequency for the QIV-specific antibody lineage with the number indicating the median value for the time point. The red point indicates the frequency of GC T_FH within CD4⁺ T cells of the LN for the time point. Donors 321-04 and 321-05 are shown in the top and bottom panels, respectively.

Extended Data Fig. 6. Bulk and single-cell TCR repertoire profiling of cT_FH and LN T_FH cells after influenza vaccination.

a–c, Network graph depicting the connections between T_FH clonal lineages in PBMCs and LN for donors: 321-04 (a), 321-08 (b) and 321-07 (c) at D0_Y1, D5_Y1, D12_Y1, D120_Y1, D0_Y2, D14_Y2, D60_Y2 and D90_Y2 time points. Node sizes correspond to the number of unique clones, and edge widths correspond to the number of clones connecting each node. d, 2D UMAP projection of total T cell dataset highlighting cells with matching paired TCR sequences to TCR data generated by scPCR of individually sorted CXCR5⁺CD38⁺ cT_FH and CXCR5⁺PD-1⁺ LN T_FH cells for donors 321-04 and 321-05. e, Frequency cells within each GEX cluster matching to TCRα or TCRβ sequences in the bulk TCR repertoire sequencing from bulk-sorted CXCR5⁺CD38⁺ cT_FH and CXCR5⁺PD-1⁺ LN T_FH cells for donors 321-04 and 321-05. TCRs were sequenced from the following combinations of donor, time point and tissue: donor 321-05: D12_Y1, D28_Y1, D60_Y1 in LN and D5_Y1 PBMCs; donor 321-04: D5_Y1 and D12_Y1 in LN and D5_Y1 PBMCs; donor 321-11: D6_Y1, D18_Y1, D21_Y1, D35_Y1 in LN. f, Morisita–Horn index of overlapping clonotypes for TCRα (left) and TCRβ (right) in bulk TCR sequencing from e.

Extended Data Fig. 7. Identification and profiling of influenza-specific T_FH clonotypes in donor 321-05.

a, Gating strategy for assessing Jurkat T_FH TCR cell line activation in co-culture experiments. b, Frequency of CD69⁺ Jurkat T cells expressing TCRs T1, T3 and T12 after co-culture with aAPCs infected with influenza PR8 for 24 hours. c, Frequency of CD69⁺ T1, T3 and T12 Jurkat T cell lines after co-culture with aAPCs transfected with plasmids expressing individual segments of the IAV genome compared to empty vector control for 24 hours. d, Frequency of CD69⁺ Jurkat T cells expressing TCRs T4, T6, T7, T9 and T10 after co-culture for 24 hours with partially HLA-matched B cells pulsed with influenza protein peptide pools. e, Frequency of CD69⁺ in T6 Jurkat cells stimulated as in d in the presence of antibodies blocking specific MHC class II molecules. f, Frequency of CD69⁺ T11 cell line co-culture with aAPCs pulsed with recombinant HA protein, PMA/ionomycin or DMSO control for 24 hours. g, PC1 scores of individual cells from the picked T_FH lineages with respect to time. h, Heatmap showing the expressions of genes corresponding to the head and tail PC1 loadings in the twelve T_FH clonal lineages.