Spatiotemporal development of the human T follicular helper cell response to Influenza vaccination

Abstract

We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.

Figure 1.. Detection of human TFH response in multiple tissues following seasonal Flu vaccination.

A) Schematic of PBMC draws and lymph node fine needle aspirates (LN) for the three donors following seasonal influenza vaccination. B) Scatter plots of cTFH (CXCR5+ CD38+) cells in the blood for donor 321–05 during year 1. Gated on CD45+ CD14- CD19- CD3+ CD4+ cells. See figures S1A-B for gating strategy. C) Frequency of ICOS+ CD71+ cTFH cells gated in B. D) Frequency of PBMC cTFH and LN TFH (CD4+ CXCR5+ PD-1+ BCL6+) and GC B cells (CD19+ IgDlo CD20hi CD38int) by time point. Shown as log10 frequency of CD4+ T cells or CD19+ B cells. E) Scatter plots of TFH (CXCR5+PD-1+) cells in LN samples. Gated on CD14- CD19- CD4+ cells. F) Frequency of lymph node CD27+ BCL6+ TFH cells gated in E.

Figure 2.. Spatial-temporal GEX and TCR repertoire profiling of T cell response to Flu vaccination.

Aggregate scGEX dataset containing N = 154,547 cells with 127,471 unique TCR clonotypes generated from PBMC and LN samples at each time point for donors 321–05 and 321–04 (See Figure 1A). 2D umap projection annotated by: A) GEX cluster T cell phenotype, B) donor origin, C) T cell type, D) tissue origin, E) study year, and F) clone size. G) Feature plots displaying select T subset markers.

Figure 3.. Identification of TFH maturation states.

Subset of the aggregate T cell dataset in figure 2 containing all cells clonally related to those in the TFH and Treg clusters. N = 15,290 cells with 11,268 unique TCR clonotypes. 2D umap projections of the TFH lineage dataset annotated by: A) GEX cluster, B) donor, C) tissue, and D) time post-immunization. E) Scatter plot of comparing the relative frequencies of germinal center B cells versus TFH cells in LN samples. Pearson correlation. F) 2D umap projection of the TFH lineage dataset annotated by TFH subset. G) Volcano plots of TFH subset-specific marker genes for pre/memory vs. IL10 TFH (left), Treg (middle), and GC TFH (right). H) Dot plot of select TFH and Treg subset-specific markers.

Figure 4.. Alterations in TFH metabolism and signaling with time.

A) GSVA of top upregulated GO terms identified by Enrichr analysis for each IL10 TFH subset relative to other TFH/Treg subsets. B) GSVA of donor 321–05 LN TFH cells ( Tregs excluded) across time points..

Figure 5.. TFH composition and phenotype are dynamic.

A) Relative frequencies of pre/memory, GC, and IL10 TFH subsets in LN samples from donor 321–05 over time. Chi-squared with p-value determined by Monte Carlo simulation. 2D umap projection of LN TFH cells ( Tregs excluded ) from all donors annotated by: B) pseudotime with selected root nodes where scale was pseudotime scale was set to zero indicated by black circles, C) TFH subset, and D) time point. E) Scatter plot of log normalized expression of TFH subset marker genes versuses pseudotime values per cell. E) Density plots of pseudotime values by TFH subset. F) Density plots of pseudotime values for 321–05 LN TFH cells for matched time points between study years. In the format (year 1 day, year 2 day), time points shown are pre (0,0), early (7,5), mid (28,28), late (60,60), final (180,120). Vertical lines indicate the median pseudotime from E) for the indicated TFH subset.

Figure 6.. Clonal TFH expansion following flu vaccination.

A) Relative frequency of TFH cells in LN samples with respect to time point. B) TFH clonality in LN samples with respect to time point as measured by inverse D50 index. C) Scatter plot of comparing the TFH relative frequency and clonality across all time points. Pearson correlation. D-G) Network graph depicting the connections between TFH clonal lineages in PBMCs and LN for donor 321–05 D), 321–04 E), 321–08 F), and 321–07 G). Node sizes correspond to the number of clones and edge widths correspond to the number of clones connecting each node.

Figure 7.. Dynamic alteration of phenotypes in Flu-specific TFH clonal lineages.

A) Alluvial plot showing the number of cells detected in PBMCs (top) and LN (bottom) for each TFH clonal lineage picked from donor 321–05 for screening across time points. B) Alluvial plot showing the relative abundance of picked clonal lineages in bulk TCRβ sequencing from sorted cTFH and LN TFH cells. C) Frequency of CD69+ or IFNγ+ TFH1, TFH3, and TFH12 Jurkat T cells after co-culture with aAPCs pulsed with indicated IAV-derived peptides. D) Frequency of IFNγ+ TFH12 Jurkat T cells after co-culture with aAPCs transfected with plasmids containing truncated versions of HA. E) PC1 scores of individual cells from the picked TFH lineages with respect to time. F) Heatmap of pre/memory, GC, and IL10 TFH gene set module scores for the picked TFH lineages. G) Heatmap of TFH marker gene expressions for TFH clones 1 and 9. In C and D the bar height corresponds to the mean, the whiskers show the standard error of the mean, and p-values calculated by t-test.